Antioxidant Nanomedicine Protects Against Ionizing Radiation

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Antioxidant Nanomedicine Protects Against Ionizing Radiation–Induced Life-Shortening in C57BL/6J mice Chitho Feliciano, and Yukio Nagasaki ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01259 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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ACS Biomaterials Science & Engineering

Antioxidant Nanomedicine Protects Against Ionizing Radiation–Induced LifeShortening in C57BL/6J mice

Chitho P. Feliciano1, 2 and Yukio Nagasaki1, 3, 4, *

1Department

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

of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan; 2Radiation Research Center (RRC), Atomic Research Division, Philippine Nuclear Research Institute, Department of Science and Technology (PNRI – DOST), Commonwealth Avenue, Diliman, Quezon City, Philippines; 3Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan; 4Center for Research in Isotopes and Environmental Dynamics (CRiED), University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan *Correspondence: [email protected] (Y. Nagasaki)

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Abstract This study examined a new redox nanoparticle (RNPN) for its ability to protect mice from the life-shortening effect of radiation. A single dose of RNPN was injected subcutaneously into mice 3 or 24 h before whole-body irradiation. Kaplan-Meier survival analysis showed that RNPN given 24 h before whole-body irradiation protected mice against radiation-induced mortality. Histological evaluation of the bone marrow showed that the probable cause of sporadic deaths was bone marrow aplasia. Furthermore, analysis of blood biomarkers revealed abnormalities in the irradiated control group. Such symptoms/indicators of poor health were reduced in RNPN-treated mice.

Keywords: nanomedicine, redox nanoparticles, radiation protection, ionizing radiation, radiation-induced life-shortening, bone marrow, antioxidant

INTRODUCTION Accidental exposure to moderate to high acute doses of ionizing radiation, as well as long-term cumulative exposures, can cause serious injuries in many cells and to the body as a whole. It is documented that people who were exposed previously to accidental doses of radiation eventually develop disorders in their bodily functions; these are known collectively as “late-effects of radiation,” as they usually manifest later in life 1. In fact, even after several years of the Fukushima nuclear plant accident in Japan, some nuclear workers, as well as civilians who were exposed to the radiation emitted from the crippled reactor, are at high risks of developing leukemia and thyroid cancers 2, 3. With increasing numbers of radiation workers,


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as well as recurring nuclear threats and terrorism, it is very important to develop new radiation protective agents that can be used by the public. There are only two drugs, amifostine and parifermin, which have been approved by the United States Food and Drug Administration as radioprotectants 4. However, their use and efficacy possess some limitations 5. In addition, the use of other low molecular weight (LMW) drugs with antioxidant functions as radioprotective agents has proven to be ineffective owing to their poor bioavailability, rapid clearance, and some unwanted side effects when used for clinical purposes 6. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), a stable nitroxide radical compound, like other LMW compounds, possesses these same disadvantages. Previously, we developed a NH2-TEMPO-containing redox nanoparticle and called it RNPN. RNPN contains an amphiphilic block copolymer that is covalently conjugated to NH2-TEMPO at the hydrophobic segment to prevent its leakage. In aqueous media, the nitroxide radical-containing block copolymer spontaneously forms core-shell type polymeric micelles 7. By this method, we have shown successfully that a nanosized formulation containing active nitroxide radicals can circulate in the blood for significantly longer durations. This extended blood circulation is attributed to the increased particle size that results in a slower excretion rate 8. Furthermore, the size of the nanoparticles prevents their rapid uptake by cells, which otherwise may disrupt the normal redox state and cause some adverse effects. These unique characteristics are the main reasons for the high therapeutic efficacy of RNPN in vivo 7 – 12. In a previous report, we demonstrated that RNPN effectively reduced organ dysfunction and death in mice after 7.5 Gy of whole-body irradiation, a lethal dose 8. Enhanced bioavailability, in addition to its extremely low toxicity, are responsible for this high efficiency. Here, we show that RNPN can also reduce significantly the life-shortening effect of ionizing radiation in vivo. Blood biochemistry of the surviving mice (moribund) in the irradiated control



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group indicated various disorders. These radiation-induced disorders were not observed in the RNPN-treated group. Histological evaluation of mice treated with RNPN also revealed that they were protected against the development of severe bone marrow depletion and cellular death. We, therefore, recommend further evaluation of RNPN for eventual use by radiotherapy patients, nuclear workers, and radiation workers who are at risk of exposure to ionizing radiation.

MATERIALS AND METHODS

Preparation of RNPN In this study, the same RNPN were used that we produced and characterized previously. Details of the preparation technique can be found in our previous publication 8.

Animals Seven-week-old C57BL/6J female mice were purchased from Charles River Laboratory International Inc., Japan, and were used in all experiments. Mice were acclimatized for one week before use and maintained under controlled clean environmental conditions (12 h light/dark cycle) throughout the duration of the experiments. Mice were fed a sterilized rodent diet and water ad libitum. Animal experimental plans for the conduct of this study were approved by the Animal Ethics Committee of the University of Tsukuba (approval numbers: 13-417, 15-325, and 16-315).

Irradiation experiments


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After acclimatization, mice (n = 10/group) were injected subcutaneously with 200 mg/kg of RNPN 3 and 24 h before whole body irradiation (6 Gy). Controls such as: 4-aminoTEMPO (NH2-TEMPO, 60 mg/kg), 4-hydroxy-TEMPO (TEMPOL, 60 mg/kg), and the base polymer (vehicle) were also injected 3 h before irradiation. Irradiation was conducted using the MBR-152OR-3 X-ray irradiator (Hitachi Power Solutions, Ibaraki, Japan) with a dose rate of 3 Gy per minute at room temperature (25 – 27°C) with the following settings: 150 kV, 20 mA, and 330 mm focus distance, with 0.5 mm aluminum + 0.1 mm copper filters. For irradiation, mice were placed inside a plastic holder that rotated during irradiation. Mice that received no treatment were kept under the same conditions and used as the healthy control group. Overall health, weight, and survival of the mice were monitored daily for 30 days, and up to 555 days post irradiation to cover the documental life expectancy of the C57BL/6J mice. After 30 days, cumulative weight loss was calculated and analyzed for statistical significance. Kaplan-Meier survival curve analysis was performed for comparison.

Blood biochemistry and hematological evaluation Blood samples were obtained from mice that survived or were moribund at the end of the trial period (> 1.5 years’ post-irradiation). Mice were sacrificed by overdose of isoflurane through inhalation. Whole blood was collected using heparinized syringes via cardiac puncture and centrifuged immediately for 2 min to separate the plasma, then stored on ice until analysis. The following blood enzymes and metabolites were measured using the FUJI DRI-CHEM 7000V analyzer (Fujifilm, Tokyo, Japan): glucose (GLU), lactate dehydrogenase (LDH), total bilirubin (TBIL), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE). Other basic hematological parameters (i.e., white blood cells (WBC) and hemoglobin (HGB)) were



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analyzed using the Nihon Kohden, Tokyo, Japan MEK-6458 blood counter. Results were compared with those of untreated healthy mice as control.

Histological examination Examinations of the vital organs and bones collected from surviving and moribund mice were also conducted. Organs and tissues were placed immediately in neutral buffered formalin solution for 24-48 h, sliced (5 µm thickness), and subsequently stained with hematoxylin and eosin for microscopic examination.

Statistical analysis Data were analyzed for statistical significance using a one-way analysis of variance with the Tukey-Kramer post-hoc test. Values with P < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

On the basis of our previous results on blood circulation tendencies and the bioavailability of RNPN in mice, we chose to inject this material subcutaneously 3 and 24 h before irradiation 8. The sub-lethal radiation dose of 6 Gy was chosen based on our previous results with C57BL/6J mice (data not shown). Mice injected with RNPN at 3 and 24 h before 6 Gy irradiation, as well as the control groups, were monitored daily for 30 days for survival, weight loss, and overall health status. At this dose, all irradiated mice suffered observable radiation-induced damage, such as weight loss and a decrease in their overall health status, during the first 30 days, but most were able to recover and survived the critical 30-day


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monitoring period. At the end of the 30-day period, cumulative changes in baseline body weight of the treated and control groups were calculated as percentages and compared. Only mice injected with RNPN and the LMW compound, NH2-TEMPO, recovered their healthy status and gained weight. Irradiated control mice and the irradiated groups injected with vehicle or LMW TEMPOL exhibited significant weight loss at day 30. The percentage of irradiated mice that recovered after receiving RNPN was significantly greater than that of the irradiated control group (Fig. 1). After the first 30 days, the mice were maintained for over 1.5 years (555 days), covering the reported lifespan for this strain of mice 13. Figure 2 shows the Kaplan-Meier survival curves of C57BL/6J mice post-irradiation. The irradiated control group and the group injected with vehicle all suffered sporadic deaths, or were found moribund within the monitoring period. Other groups recorded the following cumulative survival rates: 10% for TEMPOL, 30% for NH2TEMPO, and 20% for RNPN when given 3 h before irradiation. However, a significantly improved survival rate (70%) was recorded for mice given RNPN 24 h before irradiation. These results indicate the positive protective effects of RNPN against radiation-induced lifeshortening. All mice in the healthy control unirradiated group remained alive during the entire duration of the experiment. To determine the probable causes of death of the irradiated mice, blood biochemistry was performed, and key enzymes and metabolites were measured. The levels of enzymes associated with organ dysfunctions and diseases were significantly elevated in the irradiated group as compared with that in the unirradiated control mice (Fig. 3). In contrast, blood biomarkers of diseases (TBIL, AST, and ALT) did not increase when mice were treated with the redox nanoparticles 24 h before irradiation. Similarly, the levels of other blood enzymes (GLU, LDH, ALP, and CRE) in the RNPN-treated mice were similar to those in the healthy controls.



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Increases in the levels of AST and ALT are associated with hepatic diseases, and increases in TBIL indicate the occurrence of jaundice. These enzymes and metabolites, which were significantly elevated in the irradiated control mice, indicate abnormalities in organ functions attributable to the damage caused by exposure to ionizing radiation. Other basic blood parameters were also measured (i.e., WBC, RBC, and HGB). The results showed no significant differences among the treatment groups, except for HGB levels of the irradiated control, which were significantly lower than that in the non-irradiated group (Table 1). Studies on the biological effects of ionizing radiation have revealed the association between radiation exposure and bone marrow dysfunction resulting in multiple diseases (e.g., leukemia and aplastic anemia) 1, 14 – 18. Therefore, we examined the bone marrow of irradiated mice and compared it with that in the RNPN-treated group. Figure 4 shows the histology of bone marrow collected from the different treatment groups. The results show that the redox nanoparticles protected bone marrow against radiation-induced cell death. Depletion of bone marrow cells in irradiated control mice was very evident, while numerous healthy cells were present in the RNPN-treated group. These results indicated that radiation-induced organ dysfunctions, including abnormalities in the bone marrow tissue, caused the death of irradiated mice. Moreover, incidence of malignant tumors in the mice were observed.

CONCLUSIONS Overall, these results demonstrate that RNPN, when given via subcutaneous injection 24 h before exposure to ionizing irradiation, can significantly protect mice against the lifeshortening effect of radiation. The nanoparticles achieve this by protecting bone marrow, as well as other organs, against radiation-induced damage. This indicates that RNPN is a candidate


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novel nanomedicine for use as a radiation protectant. In particular, RNPN can prevent the late effects of radiation exposure that may result in the eventual death of the subject.

AUTHOR CONTRIBUTIONS C.P.F. conceived, designed, and performed all experiments, analyzed, summarized, and interpreted the data, and is the lead author of the paper. Y.N. conceptualized the design and synthesis of the nanoparticles, analyzed and interpreted the data, edited the paper, and supervised the entire project.

CONFLICT OF INTEREST None.

ACKNOWLEDGEMENT This work was supported by a Grant-in-Aid for Scientific Research S (25220203) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the World Premier

International

Research

Center

Initiative

(WPI

Initiative)

on

Materials

Nanoarchitectonics. C.P.F. would like to thank the Philippine Nuclear Research Institute, Department of Science and Technology (PNRI-DOST) of the Republic of the Philippines, and the Japanese Government, for the Ph.D. scholarship he received under the MEXT (Monbukagakusho) Scholarship Program. The authors would like to thank Professors Koji Tsuboi and Kenshi Suzuki of the Proton Medical Research Center, University of Tsukuba, for the use of the X-ray irradiator.



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Figure legends Figure 1. Cumulative change in baseline body weights of irradiated mice 30 days postirradiation. Mice were injected subcutaneously with 200 mg/kg of RNPN 3 or 24 h before irradiation (6 Gy). Controls such as: NH2-TEMPO (60 mg/kg), TEMPOL (60 mg/kg), and the base polymer (vehicle) were also injected 3 h before irradiation. Treated mice were monitored closely during the first 30 days post-irradiation for weight loss/gain. Percentages of the cumulative change in baseline body weights were calculated on day 30. Treatment groups were: a, irradiated control; b, vehicle; c, NH2-TEMPO; d, TEMPOL; e, RNPN at 3 h; f, RNPN at 24 h. Confidence limits (95%) for each survival curve are shown (red dashed lines). n = 10/group. RNPN, nitroxide radical-containing redox nanoparticles; NH2-TEMPO, 4-aminoTEMPO; TEMPOL, 4-hydroxy-TEMPO. *P < 0.05 and **P < 0.01 indicate significant differences versus the irradiated group.

Figure 2. Protective effects of RNPN against ionizing radiation-induced life-shortening in C57BL/6J mice. Mice were injected subcutaneously with 200 mg/kg of RNPN 3 or 24 h before irradiation (6 Gy). Controls such as: NH2-TEMPO (60 mg/kg), TEMPOL (60 mg/kg), and the base polymer (vehicle) were also injected 3 h before irradiation. Treated mice were monitored closely during the first 30 days post-irradiation and up to 555 days, covering the reported lifespan of C57BL/6J mice. Kaplan-Meier survival curves after irradiation: a, irradiated control; b, vehicle; c, NH2-TEMPO; d, TEMPOL; e, RNPN at 3 h; f, RNPN at 24 h. Confidence limits (95%) for each survival curve are shown (red dashed lines). n = 10/group. RNPN, nitroxide radical-containing redox nanoparticles; NH2-TEMPO, 4-amino-TEMPO; TEMPOL, 4 -hydroxy-TEMPO.


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Figure 3. Blood biochemistry of irradiated C57BL/6J mice. Blood samples were obtained from mice that survived or were found moribund at the end of the trial period (555 days after 6 Gy irradiation). Glucose (GLU), lactate dehydrogenase (LDH) activity, total bilirubin (TBIL),

alkaline

phosphatase

(ALP),

aspartate

aminotransferase

(AST),

alanine

aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) levels are shown. Results were compared with those of untreated healthy mice as control. Data are expressed as means ± SEM (n = 3). Significant differences between the treatment and control groups were determined using one-way analysis of variance with the Tukey-Kramer post-hoc test. Treatment groups were: no treatment (control), 6 Gy (irradiated), and 6 Gy + 200 mg/kg RNPN injected subcutaneously 24 h before irradiation. RNPN, nitroxide radical-containing redox nanoparticles.

Figure 4. Redox nanoparticles protect against bone marrow depletion and death in C57BL/6J mice. Bones were collected from mice that survived or were found moribund at the end of the trial period (555 days post-irradiation). Histology of: a, epiphysis of femur; b, epiphysis of tibia; c, tibia; and d, central tibia. Scale bar, 100 µm. Treatment groups: no treatment (control), 6 Gy (irradiated), and 6 Gy + 200 mg/kg RNPN injected subcutaneously 24 h before irradiation. RNPN, nitroxide radical-containing redox nanoparticles.

Table 1. Hematological analyses of irradiated C57BL/6J mice. Blood samples were obtained from mice that survived or were found moribund at the end of the trial period (555 days after 6 Gy irradiation). Data for white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) are shown. Results were compared with those of untreated healthy mice as control and



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known published values. Data are presented as means ± SEM (n = 3). Significant differences between the treatment and control groups were determined using one-way analysis of variance with the Tukey-Kramer post-hoc test. There are no significant differences among the treatment groups for all parameters measured, except for hemoglobin (HGB) vs. control (*P < 0.05). Treatment groups: no treatment (control), 6 Gy (irradiated), and 6 Gy + 200 mg/kg RNPN injected subcutaneously 24 h before irradiation. RNPN, nitroxide radical-containing redox nanoparticles.


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12. Y. Ikeda, K. Shoji, C.P. Feliciano, S. Saito, Y. Nagasaki. Antioxidative nanoparticles significantly enhance therapeutic efficacy of an antibacterial therapy against Listeria monocytogenes infection. Mol. Pharm., 2018, 15 (3): 1126-1132. 13. L.L. Peters, K.L. Svenson, R.V. Smith, Data Set/project: Peters3: Variation in Hematology Parameters over Time for C57BL/6J Control, Mouse Phenome Database at the Jackson Laboratory, 2005. Available online: http://phenome.jax.org/db/q?rtn=projects/projdet&reqprojid=179. 14. J.B. Storer, Chemical protection of the mouse against radiation-induced life shortening. Radiation Research, 1971, 47: 537-547. 15. J.R. Maisin, A. Decleve, G.B. Gerber, G. Mattelin, M. Lambiet-Collier. Chemical protection against the long-term effects of a single whole-body exposure of mice to ionizing radiation. Radiation Research, 1978, 74: 415-435. 16. J.R. Maisin, G.B. Gerber, M. Lambiet-Collier, G. Mattelin, Chemical protection against long-term effects of whole-body exposure of mice to ionizing radiation: III. The effects of fractionated exposure to C57Bl mice. Radiation Research, 1980: 82: 487-497. 17. S. Fujita, H. Kato, W.J. Schull, The LD50 associared with exposure to the atomic bombing of Hiroshima and Nagasaki. J. Radiat. Res., Supplement, 1991, 154-161. 18. S.C. Finch. Radiation-induced leukemia: Lessons from history. Best Practice & Research Clinical Haematology, 2007, 20 (1): 109-118.


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Figure 1.



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Figure 2.


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Figure 3.a – d.



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Figure 3.e – h.


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Figure 4.



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Table 1. Treatment Grps. No treatment 6 Gy 6 Gy + RNPN@24 h

WBC (10^2/µL) RBC (10^4/µL) HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

MCHC (g/dL) PLT (10^4/µL)

24 ± 8

916 ± 93

13.8 ± 1.3

42.1 ± 4.0

45.9 ± 0.4

15.1 ± 0.2

32.8 ± 0.1

80.0 ± 35.4

35 ± 13

602 ± 302

8.7 ± 3.8*

27.6 ± 11.2

47.1 ± 4.9

14.8 ± 1.1

31.4 ± 1.1

32.3 ± 16.7

16 ± 7

525 ± 151

7.9 ± 1.7

24.8 ± 5.3

47.8 ± 3.6

15.2 ± 1.1

31.8 ± 0.2

74.9 ± 26.9

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Antioxidant Nanomedicine Protects Against Ionizing Radiation–Induced LifeShortening in C57BL/6J mice Chitho P. Feliciano1, 2 and Yukio Nagasaki1, 3, 4, * 1Department

of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan; 2Radiation Research Center (RRC), Atomic Research Division, Philippine Nuclear Research Institute, Department of Science and Technology (PNRI – DOST), Commonwealth Avenue, Diliman, Quezon City, Philippines; 3Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan; 4Center for Research in Isotopes and Environmental Dynamics (CRiED), University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan *Correspondence: [email protected] (Y. Nagasaki)


Antioxidant Nanomedicine Protects Against Ionizing Radiation

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