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Evaluation of the Toxicity and Antioxidant Activity of Redox Nanoparticles in Zebrafish (Danio rerio) Embryos Long Binh Vong, Makoto Kobayashi, and Yukio Nagasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.6b00225 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Molecular Pharmaceutics

Evaluation of the Toxicity and Antioxidant Activity of Redox Nanoparticles in Zebrafish (Danio rerio) Embryos

Long Binh Vong‡, Makoto Kobayashi§ and Yukio Nagasaki*,‡,‫ۅ‬,¶

‡

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

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

Department of Molecular and Developmental Biology, Faculty of Medicine,

University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan; ‫ۅ‬

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

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

Satellite

Laboratory,

International

Center

for

Materials

Nanoarchitectonics

(WPI-MANA), National Institute for Materials Science (NIMS), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

* Corresponding author: Prof. Yukio Nagasaki, Department of Materials Science, Graduate School of Pure and Applied Sciences; Master’s School of Medical Sciences, Graduate

School

of

Comprehensive

Human

Sciences;

1 ACS Paragon Plus Environment

Satellite

Laboratory,


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WPI-MANA, NIMS, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

E-mail address: [email protected] Phone: +81-29-853-5749 Fax: +81-29-853-5749

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


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ABSTRACT Recently, we have been developing polymer and nanoparticle-based antioxidative nanotherapeutics. Our strategy is to eliminate overproduced reactive oxygen species (ROS), which are strongly related to various diseases. In order to facilitate the transition of the nanotherapeutics into clinical studies, we investigated the toxicity and antioxidant activity of our nanoparticles in a zebrafish model. In this study, zebrafish larvae were exposed to our highly ROS-scavenging nanoparticle (RNPO), which was prepared using our

original

amphiphilic

block

copolymer,

methoxy-poly(ethylene

glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)oxymethylstyrene] (MeO-PEG-b-PMOT). low-molecular-weight

When

the

larvae

(LMW)

were

exposed

to

nitroxide

10–30

mM

of

radical

(4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl; TEMPOL), all were dead after 12 h, whereas no larvae death was observed after exposure to RNPO at the same high concentrations. By staining mitochondria from the larvae, we found that LMW TEMPOL significantly induced mitochondrial dysfunction. In contrast, RNPO did not cause any significant reduction in the mitochondrial function of zebrafish larvae. It is important to reaffirm that RNPO treatment significantly enhanced survival of larvae treated with ROS inducers, confirming the antioxidant activity of RNPO. Interestingly, RNPO exposure



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induced the expression of Nrf2 target gene (gstp1) in the larvae’s intestines and livers. The results obtained in this study indicate that the antioxidative nanoparticle RNPO has great potential for clinical trials as it exhibits a potent therapeutic effect and extremely low toxicity to zebrafish embryos.

KEY WORDS: Redox nanoparticles, toxicity, reactive oxygen species, mitochondrial dysfunction, inflammation, TEMPO, polymer-antioxidant

INTRODUCTION Nanoparticle-based drug delivery systems have been widely used in pharmaceutical research and clinical settings to enhance drug efficacy and minimize its adverse effects.1,2 Versatile types of nanoparticles such as liposomes, polymeric micelles, gold nanoparticles, carbon nanotubes, dendrimers, and cyclodextrines have been employed for medical applications.3 Although these nanoscale systems modify the biodistribution of conventional drugs to significantly improve their therapeutic effect, 4 ,5 toxicity remains a significant concern in the development of successful pharmaceuticals. Unstable entrapment of drugs within nanoparticle carriers is one of the most pressing issues to solve. The entrapped drug can leak during blood circulation and spread to


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non-targeted areas, increasing the risk of adverse effects throughout the body. In addition, nanocarriers themselves often cause toxic adverse effects. For example, it was reported that the interaction of these foreign nano-materials with subcellular components stimulates the generation of free radicals that cause organelle damage, and cell death.6 In fact, several groups have reported that carbon nanotubes and metal oxides induce oxidative stress and toxicity via generation of reactive oxygen species (ROS). 7 – 9 Recently, organic nanoparticles, especially polymeric micelles composed of amphiphilic block copolymers that provide high biocompatibility, bioavailability, and safety, have been applied as high performance drug delivery vehicles in clinical trials.10–13 However, a recent report has demonstrated that at high concentration even these polymeric nanoparticles stimulate the generation of cellular ROS and pro-inflammatory cytokines.6 In light of these findings, we have been recently developing new polymeric nanoparticles, which possess antioxidant activity. We have confirmed that due to their antioxidant character, our nanoparticles can be utilized not only as simple drug vehicles but also as therapeutic nanoparticle. Herein, we present the safety data from detailed experiments on zebrafish larvae treated with our antioxidative nanoparticles, and discuss the mechanism underlying their therapeutic effects. The antioxidative nanoparticles that we developed (abbreviated as RNPO), are self-assembled polymeric micelles of the



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redox

polymer,

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methoxy-poly(ethylene

glycol)-b-poly[p-4-(2,2,6,6-tetramethylpiperidine-1-oxyl)oxymethylstyrene] (MeO-PEG-b-PMOT), possessing stable nitroxide radicals (ROS scavengers) in the hydrophobic segments as a side chain via an ether linkage (Figure 1). The nitroxide radicals within the nanoparticle structure do not react with one another due to steric hindrance, nonetheless, these strong antioxidants rapidly react with ROS and other radical species in the environment that have less steric hindrance. A key point in our strategy was to install the nitroxide radicals into the polymer backbone with a covalent linkage, thus stably locating them inside the nanoparticles. Hence, no leakage of the low-molecular-weight (LMW) antioxidants was observed. The diameter of the RNPO was approximately 40 nm, with a remarkably narrow distribution and extremely high colloidal stability owing to the PEG outer shell. RNPO has been applied in several disease models that are strongly related to oxidative stress, such as cancer and ischemia reperfusion injuries. Recently, we employed our RNPO for oral administration for inflammatory bowel diseases and colon cancer.14–17 After oral administration to mice, RNPO specifically accumulated in the inflamed and tumor tissues, avoiding internalization in healthy cells, which significantly reduced adverse effects and enhanced therapeutic efficacy.15,16 It is interesting to note that orally administered RNPO did not


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cause any significant disturbances to the population of intestinal bacteria in mice.18 In a previous study, we also investigated that combination of RNPO with doxorubicin significantly enhanced chemotherapy and suppressed its adverse toxicity by scavenging overproduced ROS.19 On the basis of these results, we concluded that our antioxidative nanoparticles are highly safe and can be used not only as therapeutic nanoparticles but also as high performance drug carriers. Nevertheless, the adequate assessment of toxicity and safety of our nanotherapeutics must be addressed for pre-clinical and clinical applications. For the past decades, the zebrafish (Danio rerio) has been widely utilized as a correlative and predictive model for evaluation of nanoparticle toxicity.20,21 In this study, we determined the potential toxicity of

RNPO

and

compared

it

with

LMW

nitroxide

radical

4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) using the zebrafish embryo model. We also evaluated the antioxidant capacity of RNPO in zebrafish larvae treated with ROS inducers.

EXPERIMENTAL SECTION



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Zebrafish. All experiments were carried out using a wild-type zebrafish strain (AB). Zebrafish embryos and larvae, obtained through natural mating, were held in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, pH 6.8–6.9) containing methylene blue (0.1 mg/L). Zebrafish embryos and larvae were maintained in the experimental facilities at the University of Tsukuba under the temperature about 28 °C and lighting of 14 h-10 h light-dark cycles. All experiments were carried out with the approval of the Animal Research Committee of the University of Tsukuba. Preparation of nanoparticles. The redox polymer MeO-PEG-b-PMOT was synthesized as described in our previous reports.14,15,17 RNPO was prepared via dialysis of MeO-PEG-b-PMOT. Fluorescent rhodamine-labeled RNPO (Rho-RNPO) was prepared via interaction between reduced TEMPO moieties of MeO-PEG-b-PMOT and rhodamine B isothiocyanate, as previously reported.14 Nanoparticle exposure process. Five days post-fertilization (5 dpf) larvae were transferred into a 6-cm plate (30 embryos/plate) containing E3 medium with a pre-determined concentration of RNPO or LMW TEMPOL. The survival of the larvae was measured under light microscope at 12-h intervals throughout the 5 d of exposure. Three independent experiments were conducted for statistical analysis.


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Mitochondrial staining. After 12 h exposure to RNPO or TEMPOL, 5 dpf zebrafish larvae were washed with fresh E3 medium and stained with Mitotracker (100 nM Red CMXRos, Thermo Fisher Scientific) for 30 min. Subsequently, the stained mitochondria were observed and analyzed using fluorescent confocal microscope (Zeiss LSM 700, Germany). Induction of oxidative stress in zebrafish larvae. To confirm the antioxidant effect of RNPO, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH, Aldrich, St. Louis, MO) or H2O2 (Wako Pure Chemical Industries, Osaka, Japan) were used to induce oxidative stress in the zebrafish larvae. Thirty larvae (5 dpf) were treated with RNPO (10 mM) 1 d prior to exposure to AAPH (10 mM or 25 mM) or H2O2 (1.4 mM). The survival of larvae was determined under light microscope after exposure to AAPH or H2O2. Three independent experiments were conducted for statistical analysis. Glutathione S-transferase ʌ1 (gstp1) expression. After treatment with LMW TEMPOL or RNPO, the expression of gstp1 was determined by whole-mount in situ hybridization, which was performed using RNA probes transcribed from pKSgstp1N as described previously. 22 Dimethyl maleate (DEM, Wako Pure Chemical Industries, Osaka, Japan) was used as a positive control for gstp1 expression. Biodistribution of RNPO in zebrafish embryos. Fluorescently labeled-RNPO



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(Rho-RNPO) was used to investigate the biodistribution of RNPO in zebrafish embryos. The zebrafish embryos (3 dpf) were exposed to 1 mg/mL Rho-RNPO. At predetermined time intervals, zebrafish embryos were washed with fresh E3 medium, followed by observation under Leica AF6000 fluorescent microscope (Leica, Wetzlar, Germany).

Statistical analysis. All statistical analyses were performed using SPSS software (IBM Corp, Armonk, NY). The survival data were presented as Kaplan-Meier plots and analyzed with a log-rank test. Differences between groups were examined for statistical significance using one-way analysis of variance, followed by Tukey’s post hoc test. For all statistical analyses, three independent experiments were conducted and a value of p < 0.05 was considered significant for all statistical analyses.

RESULTS AND DISCUSSION

Exposure to RNPO did not cause zebrafish embryo death or morphology changes. The lethal toxicity of RNPO was determined and compared with that of LMW TEMPOL by counting surviving embryos during the exposure period (Figure 2). As shown in Figure 2A and B, even at lower concentrations of 1 and 3 mM, LMW TEMPOL exhibited a significant toxicity to reduce survival rate of zebrafish embryos after 3 d and 4 d of exposures, respectively. All of the zebrafish embryos were dead 10 ACS Paragon Plus Environment

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after 12 h incubation with high concentrations of LMW TEMPOL (10 and 30 mM). In contrast, few dead embryos were observed even after 5 d incubation with RNPO at the same high concentrations (Figure 2C and D). This result clearly indicates that RNPO exhibited an extremely low toxicity to zebrafish embryos compared to LMW TEMPOL.

In a previous report, cationic polymeric nanoparticles exhibited high toxicity to zebrafish embryos compared to neutral nanoparticles. For example, highly cationic poly(ethylenimine) caused an abnormal development of zebrafish embryos at concentration as low as 0.01 mg/mL, while neutral poly(N-2-hydroxypropyl) methacrylamide affected the development of zebrafish embryos at concentrations of 1 mg/mL.23 In our study, no remarkably abnormal morphological changes were observed in zebrafish embryos treated with RNPO even at a high polymer concentration (10 mg/mL; data not shown), since the redox polymer MeO-PEG-b-PMOT forms a neutral nanoparticle possessing an ROS scavenging capacity. Exposure to RNPO does not cause mitochondrial dysfunction. To further investigate the toxicity of RNPO, we evaluated the mitochondrial function of zebrafish larvae after exposure to RNPO or LMW TEMPOL. As shown in Figure 3, the healthy mitochondria in the yolk sac of zebrafish larvae were stained with Mitotracker, and were analyzed using a fluorescent microscope. The number of healthy mitochondria in the 11 ACS Paragon Plus Environment


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zebrafish larvae treated with LMW TEMPOL significantly decreased compared to that of untreated zebrafish larvae. It is interesting to note that RNPO-treated larvae did not display a significantly reduced number of healthy mitochondria compared to untreated zebrafish larvae (Figure 3). This result indicates that LMW TEMPOL induces dysfunctions in zebrafish larvae mitochondria, while RNPO does not. The mitochondrial redox signaling plays a critical function in the production of ATP and metabolites essential for cellular activities. Disruption of this redox signaling induces organelle dysfunctions, mutations, and cell death.24,25 We previously reported that LMW TEMPOL was easily internalized into healthy cells and induced the dysfunction of platelet mitochondria, while RNPO significantly improved platelet mitochondrial activity.26 It was also reported that TEMPOL was found to specially target complex I of the respiratory chain to reduce both the intracellular and mitochondrial glutathione pools, induce the impairment of oxidative phosphorylation, and decrease the mitochondrial membrane potential.27 In addition, LMW TEMPOL was facile to uptake into the bloodstream and healthy intestinal epithelial cells after oral administration to mice,14 causing adverse effects including seizure, hypotension, and agitation.17,28 On the contrary, PEGylation and the nanostructure of RNPO prevents its uptake into healthy cells,14,29,30 avoiding disruption of redox signaling by mitochondria and resulting in the


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extremely low toxicity of this nanotherapeutics. Protective effect of RNPO on oxidative stress-induced survivals rate of zebrafish embryos. It has been reported that the overproduction of ROS causes oxidative stress leading to the damage of biomolecules including DNA, proteins, and lipids. ROS-induced oxidative stress contributes to many human diseases including inflammatory bowel disease, cardiovascular disease, neurodegenerative disease, and cancer. 31,32 Recently, we confirmed the therapeutic efficiency of ROS scavenging RNPO in different disease animal models.13,20,33,34 In this study, we investigated the protective effect of RNPO on oxidative stress in zebrafish larvae treated with ROS inducers. AAPH was used to generate two potent ROS capable of inducing lipid peroxidation: an alkoxy radical (RO.) and a peroxy radical (ROO.).35 All zebrafish larvae died within 12 h and 24 h of AAPH exposure at doses of 25 mM and 10 mM, respectively. In contrast, co-treatment of AAPH with RNPO significantly increased the survival rate of zebrafish larvae compared to AAPH-treated larvae (p < 0.001; Figure 4A and B), indicating that RNPO effectively suppressed the AAPH-induced oxidative damages. We also confirmed that treatment with RNPO significantly enhanced survival in H2O2-treated larvae (p < 0.001; Figure 4C). LMW TEMPOL, the nitroxide radical in the core of RNPO, has been studied for many years in antioxidant therapy as a



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superoxide dismutase mimetic, 36 , 37 although it causes mitochondrial dysfunction leading to severe adverse effects. This result suggests that RNPO possesses various antioxidant activities against oxidative stress-induced damages in zebrafish larvae, and suppresses the possible adverse effects of the nitroxide radical TEMPOL. Exposure to RNPO induces Nrf2 target gene (gstp1) expression. As stated above, nitroxide radicals can be directly scavenge ROS and reactive radical species. We have confirmed that the direct elimination of ROS by RNPO improved cell damage from oxidative stress. The size of RNPO might suppress its internalization into healthy cell and retard its access to the healthy cell interior, ultimately, preventing mitochondrial damages. It is also possible that RNPO activates the antioxidant system protecting the zebrafish embryos from oxidative stress. Nrf2 is a key transcription factor regulating the antioxidant defense in both mammalian and fish cells.38,39 In this study, we also investigated the effect of RNPO on the expression of an Nrf 2 target gene (gstp1) by whole-mount in situ hybridization. As shown in Figure 5, RNPO exposure induced the expression of gstp1 in zebrafish larvae, although the expression level was lower than in larvae treated with DEM, a compound known to activate Nrf 2 expression (Figure 5). Interestingly, gstp1 was highly expressed in the liver and intestine of RNPO-treated larvae, which correlated well with the biodistribution of RNPO within the zebrafish


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larvae. As shown in Figure 6, RNPO was found to accumulate in liver and intestine of zebrafish larvae after exposure. This result suggests that RNPO activates Nrf2 expression in liver and intestine of zebrafish larvae enhancing the antioxidant defense system; however, the exact mechanism requires further investigation. It is reported that nitroxide radicals can activate Nrf2 system via both direct and indirect pathways.40,41 Because nitroxides can converse to the reduced hydroxylamine and the oxidized oxoammonium forms, they might serve as both anti- and pro-oxidants.36 Thus, the nitroxide radical TEMPOL might act as an electrophile to direct activate Nrf2 system. Alternatively, intracellular H2O2 production via scavenging superoxide may induce an indirect activation of Nrf2 system.40,41 In previous studies, RNPO have been studies as an antioxidative nanotherapeutics in several ROS-related disease mice models including ischemia reperfusion injuries, inflammation, and cancer.15,16,33,42,43 We have also confirmed the possible adverse effects of RNPO even for a month oral administration to mice; however, no noticeable toxicities were observed in the gastrointestinal tract or other organs in mice.15 In this study, RNPO showed an extremely low toxicity to zebrafish embryos as compared to LMW nitroxide radicals. Taken together, our results indicated that RNPO is a promising antioxidative nanotherapeutics for treating ROS-related diseases with minimizing adverse effects as



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compared to LMW conventional medications.

Conclusions In this study, we investigated the toxicity and antioxidant activity of our newly developed redox nanoparticles in zebrafish larvae. Even at high exposure concentrations, RNPO exhibited an extremely low toxicity to zebrafish larvae compared to LMW nitroxide radical. Here, we confirmed that LMW TEMPOL induced mitochondrial dysfunction in the larvae, but RNPO treatment did not, which is hypothesized to be due to the size and core-shell structure of RNPO. In addition, RNPO effectively suppressed oxidative stress in zebrafish larvae after exposure to ROS inducers. Interestingly, RNPO induced the expression of an Nrf2 target gene (gstp1), enhancing its antioxidant capacity. In summary, we developed a novel antioxidant nanoparticle, RNPO, which possesses high therapeutic efficacy with extremely low toxicity.

Acknowledgements A part of this work was supported by Grant-in-Aid for Scientific Research S (25220203) and the World Premier International Research Center Initiative (WPI 16 ACS Paragon Plus Environment

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Initiative) on Materials Nanoarchitronics of the Ministry of Education, Culture, Sports, Science and Technology of Japan. L.B. Vong would like to express his sincere appreciation for the Research Fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. The authors would like to thank Ms. Miho Takeuchi for her technical assistance of whole-mount in situ hybridization.

Figure Captions: Figure 1 PEG: biocompatibility non-immunogenicity

CH3O–(CH2CH2O)m–CH2CH2S–(CH2CH)n–H

Selfassembly

MeO-PEG-b-PMOT

Toxicity

O Nitroxide radical

N

Antioxidant

RNPO

Zebrafish embryo

TEMPO: activity anti-oxidation anti-inflammation

O

Figure 1: Redox nanoparticle (RNPO) used in this study. RNPO was prepared by self-assembly of redox polymer Meo-PEG-b-PMOT.

Figure 2



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Figure 2: Toxicity of RNPO and LMW-TEMPOL on embryo survival rate. After RNPO and TEMPOL exposure, the survival of larvae was measured under light microscope at 12-h intervals throughout the 5 d of exposure time (n = 30). (A) 1 mM; (B) 3 mM; (C) 10 mM, and (D) 30 mM (RNPO: solid lines, and TEMPOL: dash lines). The data were presented as Kaplan-Meier plots and analyzed with a log-rank test (from 3 independent experiments).

Figure 3 18 ACS Paragon Plus Environment

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Figure 3: Toxicity of RNPO and LMW-TEMPOL on mitochondrial dysfunction. Zebrafish larve mitochondria were stained with Mitotracker and analyzed using a fluorescent confocal microscope system. Scale bars: 100 ȝm. The data were expressed mean r standard deviation (n = 6) and statistical significance was examined using 1-way analysis of variance, followed by Tukey’s post hoc test (* p < 0.05).

Figure 4 19 ACS Paragon Plus Environment


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Figure 4: Antioxidant effect of RNPO against oxidative stress. After RNPO treatment with AAPH or H2O2, the survival of larvae was measured under light microscope (n = 30). (A) Dash line: AAPH 10 mM; Solid line: AAPH 10 mM + RNPO 10 mM. (B) Dash line: AAPH 25 mM; Solid line: AAPH 25 mM + RNPO 10 mM. (C) Dash line: H2O2 1.4 mM; Solid line: AAPH 10 mM + RNPO 10 mM. The data were presented as Kaplan-Meier plots and analyzed with a log-rank test (from 3 independent experiments).


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Figure 5

Figure 5: Effect of RNPO on Nrf2 target gene. Expression of gstp1 was determined by whole-mount in situ hybridization. At 5 dpf, larvae were treated with 100 ȝM DEM for 6 h or 1 mM TEMPOL or 10 mM RNPO at indication times. The arrowheads indicate positive expression of gstp1. Representative sections are shown for n = 20 larvae.

Figure 6



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0h

1h

2h

12 h

6h

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24 h

48 h Liver Intestine

Figure 6: Biodistibution of RNPO in zebrafish larvae. Zebrafish larvae were exposed to fluorescent Rho-RNPO and analyzed by fluorescent microscope. Representative sections are shown for n = 10 larvae.

References


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biocompatibility! Molecular PEG: Pharmaceutics

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CH3O–(CH2CH2O)m–CH2CH2S–(CH2CH)n–H! 1 2 3 4 5 6 7 8 9 10

Selfassembly!

MeO-PEG-b-PMOT! O Nitroxide radical!

N O

non-immunogenicity !!

Toxicity ! ! ! Antioxidant activity!

TEMPO: ! anti-oxidation! ACS Paragon Plus RNPO!Environment anti-inflammation!

Zebrafish embryo!

Danio rerio - American Chemical Society

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