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Antioxidative nanoparticles significantly enhance therapeutic efficacy of an antibacterial therapy against Listeria monocytogenes infection Yutaka Ikeda, Kazuhiro Shoji, Chitho P. Feliciano, Shinji Saito, and Yukio Nagasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00995 • Publication Date (Web): 18 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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Molecular Pharmaceutics

1

Antioxidative

nanoparticles

2

enhance therapeutic efficacy of an antibacterial

3

therapy

4

infection

5

Yutaka Ikeda1, Kazuhiro Shoji1, Chitho P. Feliciano1,2, Shinji Saito3, Yukio Nagasaki1,4

6

1

7

305-8573, Japan

8

2

9

Institute, Department of Science and Technology (PNRI-DOST), Commonwealth

against

Listeria

significantly monocytogenes

Department of Materials Science, University of Tsukuba, Tennoudai 1-1-1, Tsukuba

Biomedical Research Section, Atomic Research Division, Philippine Nuclear Research

10

Avenue, Diliman, Quezon City, Philippines 1101

11

3

12

Japan

13

4

14

Tsukuba, Ibaraki 305-8573, Japan

Faculty of Medicine, University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305-8573,

Master’s School of Medical Sciences, University of Tsukuba, Tennoudai 1-1-1,

15 16

KEYWORDS. Infection, antimicrobial, oxidative stress, antioxidant, nanoparticle

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Abstract

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Acute inflammatory conditions such as sepsis lead to fatal conditions,

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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including multiple organ failure. Several treatments such as steroidal anti-inflammatory

2

drugs are currently being investigated in order to decrease the blood cytokine level,

3

which increases remarkably. However, any of these monotherapeutic treatments are not

4

always reliable and effective, and none have drastically improved survival rates, or

5

mostly ended with failure. Reactive oxygen species (ROS) are signaling molecules

6

responsible for the production of cytokines and chemokines that can mediate

7

hyperactivation of the immune response called cytokine storm. In addition to the above

8

mentioned agents, various antioxidants have been explored for the removal of excess

9

ROS during inflammation. However, the development of low-molecular-weight (LMW)

10

antioxidants as therapeutic agents has been hampered by several issues associated with

11

toxicity, poor pharmacokinetics, low bioavailability, and rapid metabolism. In the

12

present study, we aimed to overcome these limitations through the use of antioxidative

13

nanoparticles possessing 2,2,6,6-tetramethylpipelidine-1-oxyl (TEMPO) which are

14

covalently conjugated to polymer. Although treatment with antioxidative nanoparticles

15

alone did not eliminate bacteria, combined treatment with an antibacterial agent was

16

found to significantly improve survival rate of the treated mice as compared to the

17

control group. More importantly, the antioxidative nanoparticles reduced oxidative

18

tissue injury caused by the bacterial infection. Thus, our findings highlighted the


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1

effectiveness of combination treatment with antioxidative nanoparticles and an

2

antibacterial agent to prevent severe inflammation caused by bacterial infection.

3 4

Introduction

5

Despite the development of versatile medical diagnoses, there is still an urgent need for

6

the development of efficient treatments and prevention methods for infectious diseases.

7

Case in point, around 10 million people are predicted to die every year by 2050 because

8

of a continuous rise in drug resistance (1). Pathogenic microorganisms, such as bacteria

9

and viruses, cannot always establish their infection due to the protection by immune

10

systems equipped in host organisms. Reactive oxygen species (ROS) which are

11

produced by phagocytic cells and also by activation of xanthine oxidase (2), (3) are

12

signaling molecules that promote the production of inflammatory cytokines and have

13

been reported to play important roles in immune system (4). For example,

14

myeloperoxidase has been shown to eliminate pathogens by producing hypochlorous

15

acid, which is known as one of ROS (5). NADPH oxidase, which is expressed during

16

inflammation, also kills bacteria by generating ROS (6). D-Amino acid oxidase (DAO)

17

produce hydrogen peroxide from

18

becomes more potent hypochlorite during the antibacterial action (7).

D-amino

acid, and in the presence of (MPO), it



1

However, overamplified defense and inflammatory reactions or over shooting

2

of ROS and/or reactive nitrogen species (RNS) often cause acute responses, such as

3

sepsis and septic shock (8). During these acute responses, inflammatory cells such as

4

neutrophils, macrophages, and lymphocytes produce various pro-inflammatory

5

mediators, such as tumor necrosis factor-α (TNF-α) and interleukins (9). Excessive

6

inflammation also leads to systemic damage, including increased vascular permeability,

7

tissue damage, and overactivation of complement system which causes serious organ

8

failure (10), (11).

9

Overproduction of nitric oxide (NO), which is generated through the activity of

10

inducible nitric oxide synthase (iNOS), has been reported to be associated with

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decreased vascular tonus and hypotension, which are characteristics of sepsis (12).

12

Overproduction of pro-inflammatory mediators leads to impaired innate immune

13

functions. Given that hyperactivation of the inflammatory response is a feature of sepsis,

14

drug treatments have also targeted these inflammatory mediators in addition to

15

conventional antibiotic treatments. Such treatments include treatment with steroids,

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antibodies targeting the lipid component of lipopolysaccharide or TNF-α, soluble TNF

17

receptors, and IL-1 receptor antagonists (6).

18

Given the role of ROS in severe inflammation, removal of overproduced ROS


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after infection is expected to improve therapeutic efficacy. Many studies have

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demonstrated the potency of antioxidants for the treatment of sepsis. For example,

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N-acetylcysteine (NAC), vitamin C, and vitamin E were investigated for their efficacy

4

against sepsis (13). However, the development of these low-molecular-weight (LMW)

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antioxidants has been hampered because of their poor pharmacokinetics, low

6

bioavailability, and rapid metabolism (14). Another major limitation of LMW

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antioxidants is their nonspecific internalization by normal cells because of their small

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sizes, which in turn causes disruption of normal redox reactions such as electron

9

transport chains and consequently leads to redox imbalance (15). Therefore, these LMW

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antioxidants cannot be administered in high doses to eliminate overproduced ROS.

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In recent years, we have been developing the antioxidative nanomedicine

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(radical-containing nanoparticle: RNPs) (16), which can be utilized for the treatment of

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various ROS-related diseases, such as cancer (17), (18), cerebral (19), (20),

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cardiovascular (21), and renal ischemia reperfusion injuries (22), Alzheimer’s disease

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(23), and radiation damage (24). Antioxidative nanoparticles are produced from

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antioxidative

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2,2,6,6-tetramethylpipelidine-1-oxyl (TEMPO) as a side chain of the hydrophobic

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segment (PMOT) and poly(ethylene glycol) (PEG) as the hydrophilic segment (16).

block

copolymers

(PEG-b-PMOT)


possessing


1

Nanoparticles are produced via self-assembly of PEG-b-PMOTs, and the nitroxide

2

radicals in PEG-b-PMOTs are known to catalytically scavenge ROS. The

3

pharmacokinetics and bioavailability of RNPs are very different from those of LMW

4

antioxidants because of their larger sizes (24). In contrast to conventional LMW

5

antioxidants, RNPs are less likely to be internalized by normal cells and exert minimal

6

side effects (25).

7

In this study, we evaluated the therapeutic effect of RNPs in Listeria

8

monocytogenes (L. monocytogenes)-infected mice. Although treatment with RNP alone

9

did not show significant bacterial killing activity, combined treatment with an antibiotic

10

resulted in enhancement of therapeutic efficacy of an antibacterial therapy (Fig. 1).

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Figure 1. Schematic illustration of this study

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Experimental section

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Materials

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Commercial chemicals were used without further purification. Water was


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purified using the Milli-Q system (Millipore, Molsheim, France). PEG-b-PMOTs and

2

RNPs

3

hydroxyl-TEMPO

4

copolymers (24) to produce PEG-b-PMOTs (26). RNPs were prepared via dialysis of a

5

DMF solution of PEG-b-PMOT against water.

were

prepared was

according

to

reacted

with

previously

described

methods.

PEG-b-poly(chloromethylstyrene)

Briefly, block

6 7

Animals

8

Animal studies were conducted using seven-week-old male BALB/c mice

9

weighing approximately 20 g. All mice were purchased from Charles River, Japan, Inc.

10

Mice were housed in the experimental animal facilities at the University of Tsukuba,

11

Japan in a temperature- and humidity controlled-environment with a 12-h light/12-h

12

dark cycle. All mice were fed commercial chow and sterilized water ad libitum. All

13

experiments were performed according to the Guide for the Care and Use of Laboratory

14

Animals of the University of Tsukuba (Animal Plan Number: 16-323).

15 16

Treatment of L. monocytogenes-infected mice

17

Pure cultures of Listeria monocytogenes (ATCC13932) was routine cultured in

18

tryptic soy broth (TSB) and cultured at 37 °C for 24 h. The bacterial suspension in



1

saline was adjusted to a final concentration of 1×1012 CFU/mL prior to its use. The

2

number of bacteria was measured by Mcfarland standards. The L. monocytogenes

3

solution (200 µL) was intraperitoneally injected into BALB/c mice (27). RNP,

4

amoxicillin and/or hydroxyl-TEMPO were intraperitoneally injected according to the

5

schedule described in the Figure. Numbers of mice of each treatment group were

6

described in the Figure legends. At least four mice were used for the treatment.

7 8

Evaluation of bacteria counts in organs

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Mice organs (liver, spleen, lungs and blood) were collected after 0, 1, and 2

10

days after injection with L. monocytogenes and immersed in 0.9% NaCl solution at the

11

final concentration of 0.1 g/mL. Sample organs were homogenized, serially diluted with

12

saline, and plated on tryptic soy agar plate for bacterial enumeration following the

13

standard plate count procedure (28). Plated samples were incubated at 37 °C for 24 h.

14

After incubation, colony forming units were manually counted and the corresponding

15

CFU per gram was calculated.

16 17 18

TBARS assay Mice organs (100 mg) were homogenized by the ultrasonic homogenizer in 1


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1

mL of 1.15% KCl solution. Then, 200 µL of the homogenized solution was mixed with

2

20% trichloroacetic acid solution (50 µL) and 1% thiobarbituric acid (50 µL) solution,

3

followed by heating at 37 °C for 30 min. After cooling, 250 µL of an n-butanol:pyridine

4

(15:1 v/v) mixture was added to the samples, and the mixture was vortexed thoroughly

5

(29). After centrifugation at 1470×g for 10 min, the organic layer was removed, and

6

fluorescence (532 nm) was measured on a Varioskan Flash (Thermo Scientific). The

7

concentration of malondialdehyde (MDA) which is produced from lipid peroxides and

8

reacts with thiobarbituric acid (TBA) to form MDA-TBA adduct was determined by

9

measuring fluorescence at 532 nm using tetramethoxypropane as an external standard.

10 11

Statistical analysis

12

In all experiments, statistical analyses were performed using one-way analysis of

13

variance, followed by Tukey’s post hoc test. A value of p < 0.05 was considered

14

significant for all statistical analysis.

15

Results

16

Generation of L. monocytogenes-infected model mice

17

In this study, bacterial infection model mice were prepared by intraperitoneal

18

injection of L. monocytogenes into BALB/c mice. Infection dose was determined at



1

2×1011 CFU because high mortality rate of mice and organ failures were observed at

2

this dose. Bacterial counts rapidly increased at 1 day after injection, and mice began to

3

die at 2 days after injection. Considering the rapid bacterial growth, we decided to

4

administer the treatment immediately after the bacterial injection and subsequently,

5

begin the evaluation.

6

In this study, amoxicillin was chosen as antibacterial agent against L.

7

monocytogenes because amoxicillin is one of penicillin which is used for the treatment

8

of L. monocytogenes infection. MIC value of amoxicillin determined by a dilution

9

method was 1.0 µg/mL. The model mice were administered with varying doses of

10

amoxicillin (i.p.) to determine the suitable dose. All infected mice survived by the

11

treatment with amoxicillin at doses higher than 25 mg/kg. By contrast, infected mice

12

treated with amoxicillin at 15 mg/kg showed significantly reduced survival rates,

13

although better than no drug control, indicating that the antibacterial efficacy was not

14

sufficient at this dosage (Fig. 2). Therefore, mice were administered amoxicillin (i.p.) at

15

15 mg/kg in subsequent experiments. Administration of RNPs alone did not exert any

16

therapeutic effect in infected mice even at a high dose (200 mg/kg) (data not shown).

17

This is consistent with our previous in vivo findings, which showed that RNPs did not

18

directly affect the bacteria in the coronal mucosa (26). Thus, treatment with


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1

antioxidative nanoparticles alone is not directly effective against L. monocytogenes

2

infection. Based on these results, we next investigated whether the therapeutic efficacy

3

of amoxicillin at 15 mg/kg will be improved when injected in combination with RNPs.

4 5

Figure 2. Survival rates of L. monocytogenes-infected mice at different doses of amoxicillin.

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Circle: amoxicillin at 25 mg/kg; square: amoxicillin at 15 mg/kg; triangle: saline solution (n=4).

7 8

Treatment of L. monocytogenes-infected mice

9

As mentioned above, RNPs alone did not exert any therapeutic effect in the infected

10

mice. Thus, we investigated the therapeutic effect of amoxicillin in combination with



1

RNPs. Figure 3 shows the procedure for the combination treatment. RNPs (200 mg/kg)

2

were intraperitoneally injected twice (on the same day and one day after injection with L.

3

monocytogenes). Amoxicillin (15 mg/kg) was also intraperitoneally injected daily for

4

five days. Although treatment with amoxicillin alone showed therapeutic effect, all mice

5

died after 6 days. By contrast, three out of the five mice subjected to combination

6

treatment survived for more than 6 days, demonstrating the improved therapeutic effect

7

of the combination therapy. This result clearly indicates prolonged antioxidative activity

8

of RNPs (24) protected mice from the serious damages caused by infection.

9

Bacterial counts in the blood, liver, and spleen were also determined (Fig. 4).

10

Bacterial counts in infected mice without any treatment continuously increased with

11

time. By contrast, bacterial numbers were significantly lower in mice treated with either

12

amoxicillin alone or in combination with RNPs two days after infection, thereby

13

confirming the antibacterial effect of amoxicillin. Notably, there were no significant

14

differences in bacterial counts between the mice injected with and without RNPs,

15

further validating that the RNPs did not exert bacteria-killing activity.

16


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1 2

3 4

Figure 3. Combination therapy of L. monocytogenes-infected mice. a) Treatment

5

procedure. b) Survival rates of infected mice. Diamond: uninfected mice; triangle:

6

infected mice treated with amoxicillin and RNPs; circle: infected mice without

7

treatment; square: infected mice treated with amoxicillin alone (n=5).

8



1 2

Figure 4. Bacterial counts in organs. a) blood; b) liver; c) spleen; d) lungs. Circle:

3

Infected mice without treatment; triangle: infected mice treated with amoxicillin and

4

RNPs; square: infected mice treated with amoxicillin alone. Accuracy was assessed by

5

conducting replicate analysis (n = 4).

6 7

Protection of organs from oxidative damage

8

Oxidative tissue damage is a typical feature of bacterial infection (12).

9

Progression of oxidative damage in multiple organs can be observed during sepsis.

10

Accordingly, protection of organs from oxidative damage is a promising strategy for the

11

treatment of infections. To investigate the extent of oxidative damage during infection,


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1

levels of malondialdehyde (MDA), a typical byproduct of lipid peroxidation, were

2

measured in the liver, lungs, and spleen via TBARS assay. Mice were sacrificed at 1 day

3

after injection with L. monocytogenes. A significant increase in MDA levels after

4

infection was confirmed, demonstrating the role of oxidative mechanisms in bacterial

5

infection-induced tissue damage. Importantly, MDA levels in organs were lower in L.

6

monocytogenes-infected

7

nanoparticles (RNPs, 200 mg/kg). As shown in Fig. 5, MDA levels in all organs

8

investigated in this study (liver, lungs, and spleen) were significantly lower in infected

9

mice treated with RNPs than in untreated mice. These results clearly suggested that the

10

antioxidative nanoparticles exerted a protective effect against oxidative damage caused

11

by bacterial infection, although treatment with RNPs alone did not directly kill the

12

bacteria.

mice

intraperitoneally

administered

with antioxidative

13

14 15

Figure 5. MDA levels in various organs. a) Liver; b) lungs; c) spleen. Accuracy was



1

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assessed by conducting replicate analysis (n = 4). *P

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