Combination Treatment of Murine Colon Cancer with Doxorubicin and

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Combination treatment of murine colon cancer with doxorubicin and redox nanoparticles Long Binh Vong, and Yukio Nagasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00676 • Publication Date (Web): 25 Nov 2015 Downloaded from http://pubs.acs.org on November 30, 2015

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Molecular Pharmaceutics

Combination treatment of murine colon cancer with doxorubicin and redox nanoparticles

1 2 3

Long Binh Vong‡ and Yukio Nagasaki*,‡,§,ǁ

4 5

6

‡

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University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

8

§

9

Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan

Department of Materials Science, Graduate School of Pure and Applied Sciences,

Master's School of Medical Sciences, Graduate School of Comprehensive Human

10

ǁ

11

(WPI-MANA), National Institute for Materials Science (NIMS), University of Tsukuba,

12

1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

Satellite

Laboratory,

International

Center

for

Materials

1 ACS Paragon Plus Environment

Nanoarchitectonics


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*Corresponding author: Prof. Yukio Nagasaki, Department of Materials Science,

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Graduate School of Pure and Applied Sciences; Master’s School of Medical Sciences,

3

Graduate

4

WPI-MANA, NIMS, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki

5

305-8573, Japan

School

of

Comprehensive

Human

Sciences;

6

7

E-mail address: [email protected]

8

Phone: +81 29-853-5749

9

Fax: +81 29-853-5749

10

11 12

Disclosure of Potential Conflicts of Interest The authors have no competing financial interests to declare.


Satellite

Laboratory,

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ABSTRACT

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Conventional chemotherapeutic drugs such as doxorubicin (DOX) are associated with

3

severe adverse effects such as cardiac, hepatic, and gastrointestinal (GI) toxicities.

4

Excessive production of reactive oxygen species (ROS) was reported to be one of the

5

main mechanisms underlying these severe adverse effects. Recently, we have developed

6

2 types of novel redox nanoparticles (RNPs) including pH-sensitive redox nanoparticle

7

(RNPN) and pH-insensitive redox nanoparticle (RNPO), which effectively scavenge

8

overproduced ROS in inflamed and cancerous tissues. In this study, we investigated the

9

effects of these RNPs on DOX-induced adverse effects during cancer chemotherapy.

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The DOX-induced body weight loss was significantly attenuated in the mice treated

11

with RNPs, particularly pH-insensitive RNPO. We also found that cardiac ROS levels in

12

the DOX-treated mice were dramatically decreased by treatment with RNPs, resulting

13

in the reversal of cardiac damage, as confirmed by both plasma cardiac biomarkers and

14

histological analysis. It was interesting to notice that during co-treatment with DOX

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and RNPs, the DOX uptake was significantly enhanced in the cancer cells, but not in

16

healthy aortic endothelial cells in vitro. Treatment with RNPs also improved anticancer

17

efficacy of DOX in the colitis-associated colon cancer model mice in vivo. On the basis

18

of these results, a combination of the novel antioxidative nanotherapeutics (RNPs) with



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conventional anticancer drugs seems to be a robust strategy for well-tolerated

2

anticancer therapy.

3

4

KEY WORDS: Redox Nanotherapeutics, Combination Therapy, Drug Resistance,

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Adverse Effect, Chemotherapy, Reactive Oxygen Species

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7

INTRODUCTION

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Doxorubicin (DOX), an anthracycline chemotherapeutic agent, has been used for

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the treatment of a variety of human cancers for over 30 years. The therapeutic activity

10

of DOX is mediated by its intercalation into DNA, whereby it inhibits topoisomerase II,

11

and prevents DNA and RNA synthesis.1 In addition, DOX generates reactive oxygen

12

species (ROS), inducing apoptosis in cancer cells. 2 Despite the strong anticancer

13

activity and approval by the US Food and Drug Administration, DOX causes severe

14

adverse effects in most of the major organs, especially in the heart; this problem limits

15

the treatment dose.3,4 Increased oxidative stress due to DOX administration is also

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considered as the classical mechanism of its adverse effects. In addition, repeated

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administration of DOX may induce drug resistance in cancer cells.5 To alleviate the

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DOX-induced adverse effects, antioxidants such as resveratrol, curcumin, and


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N-acetylcysteine have been used as scavengers of ROS upregulated by DOX. 6 – 8

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Although these low-molecular-weight (LMW) antioxidants have been effective in vitro,

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they have failed in clinical trials because of low bioavailability, nonspecific distribution,

4

and instability in vivo environments.

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Recently, we have been developed 2 types of redox nanoparticles (RNPs) -

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pH-sensitive redox nanoparticle (RNPN) and pH-insensitive redox nanoparticle (RNPO),

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which effectively scavenge overproduced ROS in inflamed and cancerous tissues.9,10

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These RNPs were prepared via self-assembly of an amphiphilic block copolymer

9

possessing nitroxide radicals (ROS scavengers) at the side chains of the hydrophobic

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segment. In previous study, we have confirmed that the RNPs show highly dispersible

11

and biocompatible properties with long half-life in the circulation as compared to the

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LMW nitroxide radicals.11,12 In particular, the pH-insensitive RNPO shows a longer

13

blood circulation tendency compared with pH-sensitive RNPN due to the stable

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hydrophobic core of RNPO.11,12 These RNPs have been studied as a possible treatment of

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the oxidative stress injuries and some cancers. 11 – 17 For example, intravenous

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administration of the pH-sensitive RNPN works effectively in acute renal injury and

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cerebral ischemia-reperfusion because it disintegrates in acidic environments of the

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diseased site via protonation of amino groups in the core of the pH-sensitive RNPN.11,13



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On the other hand, orally administered pH-insensitive RNPO effectively scavenges ROS

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in the inflamed colon, thus reduce inflammation in the mice with colitis and inhibit the

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development of colitis-associated colon cancer (CAC) due to the specific accumulation

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of pH-insensitive RNPO in the colon mucosa without absorption into the bloodstream

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via the mesentery.15–17

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In this work, we studied the effects of the pH-sensitive RNPN and pH-insensitive

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RNPO in combination with DOX treatment in terms of both therapeutic efficacies and

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DOX-induced adverse effects in vitro and in vivo as compared to control non-nitroxide

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radical nanoparticle (nRNP).

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EXPERIMENTAL SECTION

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Preparation and characterization of RNPs and nRNP. The pH-sensitive RNPN

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and pH-insensitive RNPO were prepared from self-assembling MeO-PEG-b-PMNT and

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MeO-PEG-b-PMOT block copolymer, respectively, as described previously.11,15

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Control nanoparticle without nitroxide radical (nRNP) was prepared from

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MeO-PEG-b-PCMS block copolymer.15 Molecular formula of these block copolymers

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are shown in Scheme 1. The size of prepared RNPs and nRNP are approximately 40 nm

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in diameter (Figure 1A) using light dynamic light scattering (DLS) measurements. The


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pH-sensitive characteristics of RNPs is evaluated using DLS and electron spin resonance

2

measurements as shown in Figure 1B.

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Cell lines and cultures. The mouse colorectal carcinoma cell (C-26) and normal

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bovine aortic endothelial cell (BAEC) were purchased from Riken BioResource Center

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(cat. # RCB2657, Riken Tsukuba Institute, Ibaraki, Japan) and JCRB Cell Bank (cat. #

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JCRB0099, National Institute of Biomedical Innovation, Osaka, Japan), respectively.

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These cells were grown in Dulbecco’s modified Eagle’s medium (DMEM;

8

Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Sigma-Aldrich, St.

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Louis, MO), and 1% antibiotics (penicillin/streptomycin/neomycin; Invitrogen,

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Carlsbad, CA) in a humidified atmosphere containing 5% of CO2 at 37 °C.

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Cellular uptake of DOX in vitro. Colon cancer C-26 cells and normal BAEC cells

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were seeded in glass plates at 5 × 104 cells/well. After 2 d of culturing, the DMEM was

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replaced with fresh media, and the DOX (final concentration 0.5 µM, Wako Pure

14

Chemical Industries, Osaka, Japan) and RNPs solutions (100 µg/mL final

15

concentration) were added to the medium. Hoechst 33342 (Invitrogen) was added 15

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min before imaging in order to stain the nuclei. The cellular uptake of DOX was

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analyzed using a fluorescent confocal microscopy system (Zeiss LSM 700, Carl Zeiss

18

Microscopy GmbH, Jena, Germany) with oil immersion at 63× magnification.



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The animals and experimental design of DOX-induced adverse effects. All

2

experiments were performed on 7- to 8-week-old male ICR mice (32–35 g body

3

weight) purchased from Charles River Japan, Inc. (Yokohama, Japan). The mice were

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maintained at the experimental animal facilities of the University of Tsukuba at

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controlled temperature (23 ± 1 °C), humidity (50 ± 5%) and lighting (12 h light-dark

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cycle). The animals were given free access to food and water. All experiments were

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performed in accordance with the Regulations for Animal Experiments of the

8

University of Tsukuba and the Fundamental Guideline for Proper Conduct of Animal

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Experiments and Related Activities at Academic Research Institutions under the

10

jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of

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Japan (the animal experimental plan number #13-229).

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The mice were injected intraperitoneally with a single dose DOX (12.5 mg/kg body

13

weight) to induce adverse effects. RNPs and nRNP (100 mg/kg body weight) were

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injected intravenously 3 times (once a day) starting on day 0 (Figure 2A). After 7 d of

15

treatment, the mice were euthanized to evaluate the efficacy of RNPs against the

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adverse effects of DOX.

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Histological assessment. After the mice were sacrificed, the major organs (heart,

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liver, kidneys, spleen, and small intestine) were weighted and fixed in 4% (v/v)


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buffered formalin (Wako Pure Chemical Industries, Osaka, Japan) for 1 d and in 70%

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(v/v) alcohol (Wako Pure Chemical Industries, Osaka, Japan) for 2 d prior to paraffin

3

embedding. Then, 7-µm-thick sections of these tissues were prepared and stained with

4

hematoxylin and eosin (H&E). Histological features were examined under the light

5

microscope.

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The superoxide assay. The superoxide measurement was evaluated as previous

7

reports.15,18,19 Briefly, the hearts were collected immediately after mice sacrifice and

8

were homogenized in cold PBS. After centrifugation for 15 min at 15,000 rpm 4 °C, the

9

supernatants were collected. To determine superoxide production, supernatants (100

10

µL) were added to well of a 96-well black plate (NUNC) containing 100 µM

11

dihydroethidium (DHE; Wako Pure Chemical Industries, Osaka, Japan), followed by

12

incubation at 37 °C for 30 min. The fluorescence intensity of each well was measured at

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the excitation wavelength of 530 nm and emission wavelength of 620 nm. DHE alone

14

served as a blank control. The superoxide values, from which the blank value was

15

subtracted, were expressed as intensity per mg of protein. The superoxide level of the

16

control group was standardized to 100%.

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Induction of CAC model in mice. CAC model mice were induced by

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intraperitoneal injection of 10 mg/kg body weight of azoxymethane (AOM,



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Sigma-Aldrich, St. Louis, MO) followed by 2 cycles of 7 d of 3% dextran sodium

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sulfate (DSS, 5,000 Daltons; Wako Pure Chemical Industries, Osaka, Japan) in the

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drinking water. A video endoscopy system (TESALA AVS, Olympus, Tokyo, Japan)

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and a previously described tumor scoring system were used to evaluate the tumor

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development in mouse colons.20 DOX (5 mg/kg) was intravenously injected to mice

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once/week, while RNPO (2.5 mg/mL) was given to mice in free drinking water from

7

day 35, and the treatments were stopped on day 70.

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Statistical analysis. All data were expressed as mean ± standard deviation (SD) or

9

standard error of the mean (SEM). Differences between groups were examined for

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statistical significance using Student’s t test and one-way analysis of variance, followed

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by Turkey’s post hoc test (SPSS software; IBM Corp, Armonk, NY). Differences with a

12

P value < 0.05 were considered significant in all statistical tests.

13

RESULTS AND DISCUSSION

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RNPs suppressed DOX-induced adverse effects. It is generally known that DOX

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administration causes severe toxicities to many organs due to ROS generation. In our

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previous studies, RNPs exhibited a high ROS scavenging activity both in vitro and in

17

vivo conditions.11,21,22 In order to investigate the effects of RNPs on DOX toxicities, 10 ACS Paragon Plus Environment

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RNPs were intravenously injected to DOX-treated mice (Figure 2A). As shown in

2

Figure 2B, the DOX-treated mice exhibited a significant body weight loss. DOX

3

administration also caused a significant weight loss of internal organs in comparison

4

with untreated mice (Table 1). On the other hand, mice co-treated with DOX and RNPs,

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particularly with the pH-insensitive RNPO, showed remarkable attenuation of the

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weight loss of the entire body and of internal organs compared to the DOX-treated mice.

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DOX treatment is well known to cause cardiac and hepatic adverse effects, which raise

8

the activity of several plasma enzymes such as lactate dehydrogenase (LDH), creatine

9

phosphokinase (CPK), aspartate aminotransferase (AST), and alanine aminotransferase

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(ALT) as compared to healthy mice. As shown in Table 2, levels of these enzymes were

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significantly lower in the combination with DOX and RNPs-treated mice.

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Furthermore, the ROS levels in the heart were significantly increased in the

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DOX-treated mice, indicating that DOX treatment caused the oxidative stress in the

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heart and resulted in the cardiac toxicity. In contrast, the cardiac ROS levels were

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significantly decreased by co-treatment with RNPs (Figure 2C), indicating the ROS

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scavenging capacity of systemically administered RNPs. This is again that

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pH-insensitive RNPO showed higher performance compared with pH-sensitive RNPN.

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In histological analyses, we observed the disorganized myofibrils and vacuolization of



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the cytoplasm in heart tissues of the DOX-treated mice (Figure 3A). DOX treatment

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also caused the loss of cellular boundaries and hepatic tissue structural pattern in liver

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tissue, as shown in Figure 3B. In contrast, the mice with combination treatments with

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RNPs showed a normal morphology with well-preserved cytoplasm in heart and liver

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tissues, which are similar to healthy control mice (Figure 3A and B). It was also

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reported that DOX treatment causes damage in intestinal tissues.23,24 As can be seen in

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Figure 4, the damage of intestinal tissues was reversed by intravenous administration of

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RNPs. These data clearly indicate that administration of RNPs effectively ameliorated

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DOX-induced cardiac, hepatic, and intestinal adverse effects in mice by scavenging

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overproduced ROS. DOX has been postulated to cause cardiac toxicity through

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activation of p53 protein and overproduction of ROS in cardiomyocytes, endothelial

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cells, and other non-cancerous cells.25,26 Numerous ROS scavengers have failed to

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prevent the toxicities of DOX in the clinical trials because of low biocompatibility and

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low stability in the blood circulation after administration. In addition, LMW

15

antioxidants are internalized by healthy cells and interfere with the important redox

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reactions such as electron transport chain, which stops respiratory system and causes

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severe adverse effects. This drawback prevents effective level of dose for LMW

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antioxidants. We have already shown that RNPs prevents internalization to healthy cells


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1

due to their nanoparticle size and large molecular weight, which do not disturb the

2

important redox reactions in healthy cells.27

3

It should be noted that the effect of the pH-insensitive RNPO is more clearly

4

observed as compared to the pH-sensitive RNPN. We previously reported that the

5

pH-insensitive RNPO exhibited a much longer blood circulation than pH-sensitive

6

RNPN, which might enhance its ROS scavenging efficiency to suppress the adverse

7

effects of DOX to mice. This finding also suggests that the short half-life of LMW

8

antioxidants in the bloodstream is one of the reasons for their weak effect on

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ROS-induced adverse effects. In addition, nitroxide radicals in the core of RNPs are

10

covalently conjugated to the polymer chains, and this arrangement prevents the leakage

11

and internalization of free active ingredients into healthy cells, suggesting low adverse

12

effects of these nanotherapeutics.

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RNPs enhanced uptake of DOX in cancer cells in vitro. As stated above, one of

14

the mechanisms underlying the antitumor effects of DOX is its intercalation into DNA

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in the nucleus. Thus, it was important to confirm cellular internalization tendency of

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DOX along with RNPs. The cellular viability and DOX uptake were analyzed during

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co-treatment of colon cancer cells (C-26) and normal endothelial cells (BAEC) with

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DOX and RNPs. As shown in Figure 5A, RNPs alone did not cause any cytotoxicity in



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1

both cell types under the present conditions. It is also obvious that the combination

2

treatment of RNPs with DOX significantly enhanced the anticancer activity of DOX

3

toward C-26 cells compared to DOX treatment alone (Figure 5A). Conversely, it is

4

rather surprising for us that cytotoxicity of DOX to normal BAEC cells was suppressed

5

by the combination treatment of DOX with RNPs (Figure 5B). One of the main

6

reactions that ROS participate in a live organism is oxidation of lipids in the cellular

7

membrane, which causes an increase in nonspecific permeation of substances. The

8

strong antioxidative effect of RNPs may attenuate such cellular membrane damages by

9

oxidative stress.

10

On the other hand, we found that the uptake of DOX in C-26 was significantly

11

improved by co-treatment with RNPs (Figure 5C and D). It was reported that excessive

12

generation of ROS upregulates the expression of P-glycoprotein (Pgp), a

13

transmembrane protein capable of effluxing many chemotherapeutic drugs out of

14

cancer cells, which is one of the major mechanisms of drug resistance phenomenon,

15

resulting in low uptake of chemotherapeutic drug in cancer cells. 28 Antioxidant

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N-acetylcysteine inhibits the expression and activity of Pgp induced by high production

17

of ROS.29 Additionally, we found that the Pgp level was decreased by incubation of a

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Pgp-overexpressing human epidermoid KB carcinoma cell line with RNPs (data not


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1

shown). The results suggested that RNPs enhanced the uptake of DOX in C-26 colon

2

cancer cells by suppressing the expression of Pgp during DOX treatment. On the basis

3

of the observed effects of combination treatments, RNPs are expected to improve

4

therapeutic efficacy and suppress adverse effects of DOX.

5

RNPs enhanced chemotherapeutic effects of DOX in vivo. Since RNPs

6

effectively suppressed the DOX-induced adverse effects and enhanced DOX uptake in

7

cancer cells, the combination of DOX with RNPs is anticipated to be an effective

8

treatment for cancer therapy. Accordingly, we next examined the efficacy of the

9

combination treatment of RNPs and DOX in vivo on a mouse model of CAC, which

10

was induced by co-treatment with AOM and DSS. We have previously reported that

11

orally administered pH-insensitive RNPO does not get absorbed into bloodstream but

12

highly accumulates in tumor tissues in the colon.17 Thus, it is interesting to investigate

13

that oral administration of RNPO works similarly to that of intravenous administration

14

in combination treatment with intravenously injected DOX. As shown in Figure 6A and

15

B, DOX alone mildly suppressed tumor growth, whereas the combination treatment

16

significantly inhibited the tumor development in the colon of mice as compared to the

17

control group and DOX-treated groups. In particularly, the survival and the lifespan

18

were remarkably improved by the combination treatment compared to the treatment



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1

with DOX only (Figure 6C). It should be noted that the lifespan of the DOX alone

2

group of mice did not increase significantly, which is interpreted by the severe adverse

3

effect to normal organs though tumor growth was suppressed as anticipated. In contrast,

4

the combination treatment with RNPO significantly improved the chemotherapeutic

5

efficiency of DOX against colon cancer development and improved the lifespan of mice,

6

probably due to the effective suppression of the adverse effects. In clinical practice, the

7

cumulative dose of DOX is limited due to its severe adverse effects, resulting in a low

8

effect of chemotherapy. The results obtained in this study indicated that administration

9

of RNPs significantly suppressed DOX-induced adverse effects in mice including

10

cardiac, hepatic, and intestinal toxicities. Therefore, the high doses of DOX can be

11

applicable in mice administrating with RNPs to achieve an effective cancer therapy

12

with low adverse effects.

13

14

CONCLUSION

15

In this study, we tested a combination treatment of novel ROS scavenging

16

nanoparticles, RNPs, with conventional chemotherapy DOX against colon cancer.

17

Administration of RNPs significantly suppressed the severe adverse effects of DOX

18

both in vivo and in vitro. In addition, RNPs enhanced the uptake of DOX in the cancer


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1

cells, not in healthy endothelial cells, which significantly improved chemotherapeutic

2

efficacy of DOX. The combination treatment was effective to inhibit colon cancer and

3

prolonged the lifespan with low adverse effects. Taken together, our results indicate that

4

the combination of antioxidative nanotherapeutics with conventional chemotherapy is a

5

promising strategy for well-tolerated anticancer therapy.

6

ACKNOWLEDGEMENTS

7

A part of this work was supported by a Grant-in-Aid for Scientific Research S

8

(25220203) and the World Premier International Research Center Initiative (WPI

9

Initiative) on Materials Nanoarchitectonics of the Ministry of Education, Culture,

10

Sports, Science and Technology (MEXT) of Japan. One of the authors, L.B. Vong,

11

would like to express his sincere appreciation for the Research Fellowship of the Japan

12

Society for the Promotion of Science (JSPS) for Young Scientists.



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1

Figure Captions:

2

Scheme 1

3 4

Scheme 1: Molecular formula of block copolymers and illustration of redox

5

nanoparticles (RNPs) and control nanoparticle without nitroxide radical (nRNP) used in

6

this study.


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1

Figure 1 A

B

RNPO

RNPN

Light scattering intensity (%)

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nRNP

120 100 80 60 40 RNP O 20

RNP N

0 1

2

10 100 1000 1 Diameter (nm)

10

100

Diameter (nm)

1000 1

10

100

1000

3

4

Diameter (nm)

5

6 pH

7

8

9

3

Figure 1: Characterization of nanoparticles. (A) The size of RNPO, RNPN, and CNP

4

are determined by dynamic light scattering (DLS, Zetasizer Nano ZS). (B) pH-sensitive

5

characteristics of RNPN and RNPO under different pH value. The light scattering

6

intensity is measured by DLS and normalization (100%) is expressed as the value

7

relative to that at pH 7.5. The electron spin resonance (ESR) is used to confirm

8

morphology of RNPs. Black ESR spectrum indicates the micelle structure of RNPO and

9

RNPN at pH 7.5. Green ESR spectrum indicates the micelle structure of RNPO at pH 2.5,

10

and red ESR spectrum indicates the disintegration of RNPN at pH 2.5.

11



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1

Figure 2

2

3

Figure 2. RNPs suppress DOX-induced adverse effects in vivo. (A) The scheme of

4

administration of the RNPs and DOX administration to mice. (B) Body weight changes

5

during treatment with RNPs and DOX. (C) Superoxide production in the heart after 7 d

6

of treatment with RNPs and DOX. The data are expressed as mean ± SEM, *P < 0.05

7

compared to the DOX group, n = 6.

8

9


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1

Figure 3

2

3

Figure 3. Administration of RNPs reverses cardiac and hepatic histological toxicity

4

induced by DOX. After 7 d of treatment, the heart and liver were collected, and

5

7-µm-thick sections were prepared. The sections were stained by hematoxylin and eosin

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(H&E) and assessed histologically. (A) Histological analysis of the heart. (B)

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Histological analysis of the liver. Representative sections are shown for n = 3 mice. The

8

scale bars are 50 µm.

9



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1

Figure 4

2

3

Figure 4. Administration of RNPs attenuates the intestinal toxicity induced by

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DOX. Intestinal histological analysis of mice after 7 d of treatment with DOX and

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RNPs. Sections were stained by H&E, and assessed histologically. Representative

6

sections are shown for n = 3 mice. The scale bars are 500 µm.

7


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1

Figure 5

2

3

Figure 5. Effects of RNPs on DOX-treated colon cancer C-26 cells and normal

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aortic endothelial BAEC cells in vitro. (A and B) Cellular viability of C-26 cells and

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BAEC cells, respectively, after 1 d of treatment with RNPs (0.5 mM) and DOX (0.5

6

µM) according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

7

(MTT) assay. The data are expressed as mean ± SD, *P < 0.05, n = 6. (C and D) Uptake

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of DOX in colon cancer C-26 cells. DOX (red) and nuclei (blue, stained with Hoechst

9

33342) were analyzed using a fluorescent confocal microscope system (Zeiss LSM

10

700) with oil immersion at 63× magnification. The scale bars are 20 µm. The data are

11

expressed as mean ± SEM, *P < 0.05 compared to the DOX group, n = 4 to 5.



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Figure 6

2

3

Figure 6. Combination effects of DOX and RNPO on AOM/DSS-induced CAC in

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mice. DOX (5 mg/kg) was intravenously injected to mice once a week, while RNPO

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(2.5 mg/mL) was administered to mice with freely drinking water starting on day 35,

6

and the treatments were stopped on day 70 (since the beginning of the AOM/DSS

7

treatment). (A) The endoscopic imaging of mice on day 140 of treatment. (B) Colonic

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tumor development profile. The data are expressed as mean ± SEM, *P < 0.05

9

compared to the DOX group, n = 6 mice. (C) The survival rate of the mice after

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treatment with DOX and RNPO, n = 6 mice.

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Table 1. Organ weight (g) after 7 d of treatment with DOX and RNPs

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Control

DOX

DOX + nRNP

DOX + RNPN

DOX + RNPO

Liver

2.152 ± 0.164 *

1.312 ± 0.090

1.365 ± 0.139

1.652 ± 0.225 *

1.767 ± 0.316 *

Heart

0.216 ± 0.020 *

0.131 ± 0.012

0.136 ± 0.0489

0.136 ± 0.010

0.174 ± 0.025 *

Spleen

0.126 ± 0.005 *

0.057 ± 0.006

0.0676 ± 0.030

0.069 ± 0.018 *

0.075 ± 0.007 *

Kidney

0.338 ± 0.021 *

0.190 ± 0.020

0.196 ± 0.069

0.215 ± 0.023 *

0.251 ± 0.028 *

* P < 0.05 compared to the DOX group, the data are expressed as mean ± SEM, n = 6. 24 ACS Paragon Plus Environment

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Table 2. Effects of RNPs on activities (U/L) of plasma LDH, CPK, AST and ALT

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enzyme in DOX-treated mice.

Control

DOX

DOX+nRNP

DOX+RNPN

DOX+RNPO

LDH

463.5 ± 45.75 *

732.17 ± 70.96

744.67 ± 78.74

764.67 ± 44.98

476.57 ± 27.22 *

CPK

461.33 ± 54.10 *

1009.17 ± 196.38

987.83 ± 210.57

467.83 ± 118.60 *

431.86 ± 55.17 *

AST

76.00 ± 5.11 *

185.50 ± 38.12

211.50 ± 61.07

159.33 ± 39.41

84.00 ± 14.06 *

ALT

24.83 ± 1.45*

76.33 ± 19.90

88.00 ± 23.26

41.40 ± 6.71 *

24.00 ± 3.77 *

3

* P < 0.05 compared to the DOX group, the data are expressed as mean ± SEM, n = 6.

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Administration of redox nanoparticles (RNPs) suppresses severe side effects of doxorubicin (DOX) by scavenging overproduced reactive oxygen species. RNPs significantly enhance the uptake of DOX in cancer cells, resulting in effective therapy against colon cancer 182x107mm (300 x 300 DPI)

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Combination Treatment of Murine Colon Cancer with Doxorubicin and

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