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[20250905 revised manuscript_maralia ver9.2_修正履歴無し.docx](https://mdr.nims.go.jp/filesets/56152510-d4db-4de6-a859-f6ddb71ffbd1/download)

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[Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), Kyoko Hayashi, Tamasa Araki, Takeshi Annoura, Yukio Nagasaki

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This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular Pharmaceutics, copyright © 2025 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.molpharmaceut.5c00419.[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[Self-Assembling Antioxidant Nanoparticles Increase Survival and Reduce Disease Symptoms of Severe Malaria](https://mdr.nims.go.jp/datasets/efe88e70-53ed-4c66-bf9f-f76808ed48af)

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Regular articleSelf-assembling Antioxidant Nanoparticles Increase Survival and Reduce Disease Symptoms of Severe MalariaToru Yoshitomi1-3, Kyoko Hayashi4, Tamasa Araki5,6, Takeshi Annoura5,7, and Yukio Nagasaki4,8-11*Affiliation:  1 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 2 Department of Materials Science and Engineering, Institute of Science Tokyo, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan 3 Master’s/Doctoral Program in Life Science Innovation (T-LSI), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan 4 Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan 5 Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 6 Research Center for Biosafety, Laboratory Animal and Pathogen Bank, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 7 Cooperative Division of Veterinary Sciences, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan 8 Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan 9 Center for Research in Radiation, Isotopes, and Earth Sciences (CRiES), University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan10 Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku Tokyo 113-0033, Japan11 High-value Biomaterials Research and Commercialization Center (HBRCC), National Taipei University of Technology, Taipei 10608, Taiwan*Corresponding Author: Yukio Nagasaki E-mail: nagasaki@ims.tsukuba.ac.jpAbstractSevere infectious diseases trigger a rapid increase in cytokines, known as a cytokine storm, accompanied by an excessive rise in reactive oxygen species (ROS), leading to severe damage to tissues and cells. While antioxidants have been used to eliminate ROS, conventional low-molecular-weight (LMW) antioxidants fail to effectively mitigate oxidative stress in infectious diseases due to rapid metabolism, excretion, and potential disruption of the redox balance within normal cells. We developed a novel self-assembling antioxidant nanoparticle, termed redox nanoparticle (RNP), designed to prevent rapid metabolism and excretion. Furthermore, due to its nanoscale size and poly(ethylene glycol) shell, RNP exhibits limited entry into normal cells, preserving the intracellular redox balance. This study evaluates the impact of RNP on oxidative stress associated with infectious diseases, utilizing a Plasmodium berghei-induced malaria model in mice. After intraperitoneal administration, RNP was absorbed into the bloodstream and remained in circulation for over 24 hours. In the malaria model, we observed that once the threshold of erythrocyte parasite infection was exceeded, ROS levels in the blood dramatically increased in untreated infected mice, causing lethal damage. The administration of LMW TEMPOL resulted in only marginal reduction in ROS, whereas RNP significantly decreased ROS levels, leading to a marked improvement in disease severity and survival. Moreover, RNP suppressed the oxidation and fragility of erythrocyte membranes caused by infection. These findings highlight the potential of RNP as a breakthrough therapeutic agent for malaria and other infectious diseases characterized by excessive oxidative stress.  Keywords:Antioxidant nanoparticles, Malaria, Infection, Oxidative stress, Reactive oxygen species1. Introduction Malaria is one of the most serious infectious diseases affecting over 80 countries, caused by a parasite that commonly infects certain mosquito species and spreads to humans through the bites of Anopheles mosquitoes.1 In 2023, there were an estimated 264 million cases of malaria worldwide, accounting for more than 619 thousand deaths.2 Some malaria patients develop severe malaria, which is characterized by one or more organ dysfunctions such as acute lung injury3, acute respiratory distress syndrome4, and acute kidney injury.5 An uncontrollable increase in various cytokines, known as a cytokine storm (CKS), occurs during malaria.6, 7 Although several anti-cytokine drugs, such as antibodies against interleukin (IL)-6, have been developed for the treatment of CKS, the expected effects are not necessarily achieved.8 Importantly, the overproduction of inflammatory cytokines is always accompanied by the excessive generation of reactive oxygen species (ROS), which further amplifies CKS and causes serious damage to tissues.9-11 Therefore, treatment with antioxidants is one of the potential strategies for suppressing CKS. Although numerous antioxidants have been developed, none have demonstrated significant efficacy against CKS.12. Most antioxidants developed to date are low-molecular-weight (LMW) compounds that are not only rapidly metabolized and excreted, but also disrupt the intracellular redox balance in normal cells.13-16 Therefore, LMW antioxidants are unable to effectively scavenge ROS that rapidly increase in the early stage of infection. Although drug delivery systems (DDS) that physically encapsulate existing antioxidants in liposomes or polymeric micelles are promising, their current major limitation lies in leakage during circulation, which leads to insufficient efficacy. Here, we report the development of a self-assembling antioxidant nanoparticle, termed redox nanoparticle (RNP), prepared via the self-assembly of amphiphilic block copolymers, methoxy-poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (MeO-PEG-b-PMNT) (see Figure 1A).17-21 Antioxidant nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyls (TEMPOs), are covalently conjugated to the hydrophobic PMNT segment, thereby completely preventing the leakage of antioxidants during blood circulation. RNP exhibits long-term circulation in the bloodstream without disrupting the intracellular redox environment, owing to its nanoscale size and biocompatible poly(ethylene glycol) (PEG) shell layers.16 Thus far, we have demonstrated that RNP treatment has significant therapeutic effects against various ROS-related diseases, such as cerebral22, 23, cardiovascular24, and renal ischemia reperfusion injuries25, 26, Alzheimer’s disease27, cancer28, 29, and radiation damage30. Our previous study focused on infection and ROS-induced damage and found that RNP administration reduced mortality in mice infected with Listeria monocytogenes.31 If RNP is also effective against infections caused by non-viral pathogens, it could become a new therapeutic modality for a variety of life-threatening infectious diseases. Although nanoparticle-based antioxidant delivery system, such as liposomes, has been investigated, they often suffer from premature antioxidant release and limited therapeutic efficacy.32 To date, the therapeutic effect of nanomedicines with covalently conjugated antioxidants has not been evaluated in malaria model mice. Therefore, in this study, we investigated whether RNP could effectively eliminate ROS and ameliorate disease outcomes in a severe malaria mouse model.2. Experimental Section2.1. MaterialsCommercially available reagents were used without further purification. Deoxyribonucleic acid sodium salt (DASS) from salmon testes, thiobarbituric acid, tetraethoxypropane, potassium ferricyanide, and dihydroethidium (DHE) were purchased from Sigma-Aldrich, Inc. (St. Louis, Missouri, USA). Dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), NaCl, and trichloroacetic acid were purchased from FUJIFILM Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Phosphate-buffered saline (PBS) was purchased from Nacalai Tesque, inc. (Kyoto, Japan) and autoclaved for sterilization before use. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and butylated hydroxytoluene were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and dissolved in PBS solution, and sterilized by filtration through a 0.22 µm pore-size syringe filter (Merck Millipore, USA). Ethylenediaminetetraacetic acid disodium salt (EDTA‐2Na) was purchased from Dojindo (Kumamoto, Japan). Giemsa’s azur eosin methylene blue solution was purchased from Merck Millipore (Darmstadt, Germany).2.2. Preparation of RNPMeO-PEG-b-PMNTs and RNP were prepared according to previously described methods 33. RNP with a diameter of 30 nm were prepared via dialysis of a DMF solution of MeO-PEG-b-PMNT against water using a dialysis membrane (MWCO: 3,500, Spectra/Por®3 Dialysis Membrane, Spectrum Laboratories, Rancho Dominguez, CA, USA). RNP solution was sterilized by filtration through a 0.22 µm pore-size syringe filter (Merck Millipore). The hydrodynamic diameter of RNP was characterized by dynamic light scattering (DLS) measurements using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Electron spin resonance (ESR) spectra of RNP and TEMPOL were recoeded using an X-band ESR spectrometer (JES-FA100; JEOL, Tokyo, Japan) at room temperature. The ESR measurements were carried out under the following conditions: frequency, 9.152 GHz; power, 10.0 mW; field, 325.9 ± 10 mT; sweep time, 0.5 min; modulation, 0.01 mT; time constant, 0.03 s.2.3. Animal experiment The animal experiment and the procedures were approved by the Institutional Animal Care and Use Committee (Approval No. 121060-II) and conducted at the National Institute of Infectious Diseases, Japan. Eight-week-old female C57BL/6J mice (CREA Japan Inc., Tokyo, Japan) were used for the experiments. The mice were housed under standard conditions in the animal facility of the National Institute of Infectious Diseases and provided with food and water ad libitum. 2.4. Preparation of the malaria model in mice and its treatment Eight-week-old female C57BL/6J mice, weighing approximately 18 g, were intraperitoneally infected with 1 × 106  Plasmodium berghei ANKA (PbA)-infected erythrocytes in 0.1 mL PBS, which were obtained from whole blood of a mouse infected with frozen stocks of PbA-infected erythrocytes.34 Percent parasitemia (percentage of erythrocytes infected with PbA) over the course of infection in mice was monitored using Giemsa-stained blood smears. Mice were divided into four groups: (1) Uninfected group with the treatment with PBS as a negative control, (2) PbA-infected group with the treatment with PBS as a positive control, (3) PbA-infected group with the treatment with RNP, and (4) PbA-infected group with the treatment with TEMPOL. The treatment started on day 4 after the infection. One hundred microliters of PBS, RNP (4.6 mg/mL in PBS), or TEMPOL (0.9 mg/mL in PBS) were intraperitoneally injected. The dosage of RNP and TEMPOL was 25 mg/kg and 5.1 mg/kg, respectively, containing the same amount of nitroxide radicals. The total administered mass of RNP was higher than that of TEMPOL because the nitroxide radicals were covalently conjugated to the PEG-PMNT polymer. The PEG shell forms a nanoparticle structure that enables prolonged circulation in the bloodstream, contributing to sustained ROS scavenging activity. The treatment continued until day 11 for the survival experiment. To evaluate the disease severity, the body temperature was measured by thermometer from day 0 to day 13.2.5. Measurement of superoxide anion in the bloodThe animals were sacrificed under anesthesia using isoflurane inhalation. After laparotomy, blood was collected from the heart using heparinized syringes, followed by centrifuging (3,500 rpm, 1,100 × g, 10 min) the blood to obtain plasma samples for the measurements of superoxide anion. To measure the level of superoxide anion in the blood, DHE was used. DHE was dissolved in DMSO to prepare stock solution at 50 mM, which was diluted with PBS 1,000 times on the day of experiment. DASS from salmon testes was dissolved in PBS to prepare stock solution at 5 mg/mL. Ten microliters of 50 μM DHE and 1 mL of DASS (5 mg/mL) were added into 8.99 mL of PBS to obtain working solution. Fifty microliters of the working solution were then added to 50 µL of plasma samples, followed by incubation for 30 min at 37 ºC. After the incubation, fluorescence intensity was measured at λex = 525 nm and λem = 640 nm using microplate reader (a DTX880 Multimode Detector, Beckman Coulter, USA).2.6. Measurements of lipid peroxidation in the erythrocytesLipid peroxidation was measured on the basis of thiobarbituric acid-reactive substance (TBARS) levels in the erythrocytes. To evaluate lipid oxidation in the erythrocytes, EDTA-2Na-containing syringes were used to collect the blood from the heart. Then, the obtained blood was centrifuged at 1,100 × g at 20 ºC for 10 min. The resulting erythrocyte pellet was washed with 0.9% NaCl solution three times and suspended in 0.9% NaCl solution. The obtained erythrocyte suspensions were diluted 50 times by MilliQ water. The diluted erythrocyte solution (50 µL) was mixed with 5 µL of 40 mM butylated hydroxytoluene, 100 µL of 30% trichloroacetic acid, and 160 µL of PBS, and kept on ice for 2 h. After centrifuging (3,500 rpm, 1,100 × g, 10 min), 200 µL of supernatant was collected and added to the mixture of 15 µL of 0.1 M EDTA-2Na solution and 50 µL of trichloroacetic acid-thiobarbituric acid (1.0% [wt/vol]). The mixture was heated at 90 ºC for 15 min and then cooled at room temperature. The absorbance of the supernatant was measured at 532 nm to measure the TBARS level. Tetraethoxypropane was used as the standard sample 35. 2.7. Measurement of osmotic fragility To assess the vulnerability of the erythrocytes in the PbA-infected mice, an osmotic fragility test was conducted 33. For the evaluation of osmotic fragility of the erythrocytes, EDTA-2Na syringes were used to collect blood from the heart. Then, the obtained blood was centrifuged at 1,100 × g at 20 ºC for 10 min. The obtained pellet was washed with 0.9% NaCl solution three times and suspended in 0.9% NaCl solution. Then, 10 µL of the erythrocyte solution and 190 µL of varying concentrations of NaCl solution were mixed and incubated for 5 min at room temperature, followed by centrifugation at 3,000 × g for 5 min. One hundred fifty microliters of the supernatant were transferred to a 96-well plate and measured using the microplate reader at an absorbance of 532 nm. The absorbances at 0.9% NaCl concentration was defined as 0% hemolysis, while the absorbance at 0.2% NaCl concentratoin was defined as 100% of hemolysis. The fragility curve was constructed by plotting the NaCl concentration versus the rate of hemolysis.2.8. Quantification of plasma nitroxide radicals using ESR.     Plasma levels of RNP and TEMPOL were measured as a proxy for systemic antioxidant capacity. One hundred microliters of RNP (33 mg/mL in PBS), or TEMPOL (6.5 mg/mL in PBS) were intraperitoneally injected to uninfected mice. The dose of RNP and TEMPOL were 180 mg/kg and 35.7 mg/kg, respectively, which contained the same amount of nitroxide radicals. Blood samples were collected from the heart using a heparinized syringe at 0.5, 1, 4, and 24 h post-intraperitoneal (i.p.) administration. The ESR signal intensities in plasma samples were measured by X-band ESR spectrometer at room temperature. For ESR measurements at 0 min, 10 μL of RNP (33 mg/mL in PBS) and TEMPOL (6.5 mg/mL in PBS) was added to the blood (150 μL), assuming that the total blood volume in mice is 80 mL/kg.36 The total amount of drug (nitroxide radicals + hydroxyamines) in plasma samples was estimated using X-band ESR measurements after the addition of potassium ferricyanide at the final concentration of 2 mM. The ESR measurements were carried out under the following conditions: frequency, 9.152 GHz; power, 10.0 mW; field, 325.9 ± 10 mT; sweep time, 0.5 min; modulation, 0.01 mT; time constant, 0.03 s.2.9. Statistical analysisStatistical analyses of Student’s t-test and one-way ANOVA with Tukey’s post hoc test were performed using Kaleida Graph 4.5 J (Synergy Software, USA). Kaplan-Meier survival analysis was performed with GraphPad Prism 10 software (GraphPad Software Inc., San Diego, CA, USA). Differences with a value of p < 0.05 were considered statistically significant. 3. Results and discussion3.1. Characterization of RNPAs previously mentioned, LMW antioxidants are not only rapidly metabolized and excreted but also disrupt the redox balance in normal cells, causing side effects that outweigh their anticipated antioxidant benefits. While DDS that physically encapsulate antioxidants in nanoparticles have been explored to regulate their excretion rate and distribution in the body, their effectiveness remains limited due to premature drug leakage during delivery, resulting in minimal therapeutic benefits. To address these challenges, we have developed a system in which antioxidant molecules are covalently conjugated with amphiphilic block polymers, which self-assemble into micelles in aqueous media and effectively prevent leakage. This system forms nanoparticles measuring tens of nanometers, making them less likely to be internalized by normal cells. Furthermore, even if internalized by cells, they do not damage essential organelles such as the endoplasmic reticulum and mitochondria. Here, we employed TEMPO as an antioxidant. TEMPO, characterized by a tetramethyl group surrounding its nitroxide radical, is recognized as a stable radical.37 However, it is also considered one of the most potent antioxidants due to its ability to rapidly react with and neutralize ROS.20, 21 As shown in Figure 1A, the molecular weights of the PEG and PMNT in the block copolymer (MeO-PEG-b-PMNT) synthesized in this study were 5,000 and 7,600, respectively. The polymeric micelles, RNP, obtained by dialyzing a DMF solution of MeO-PEG-b-PMNT against water, had an average diameter of approximately 30 nm, as determined by DLS measurements (Figure 1B). TEMPO possesses an unpaired electron, making it detectable via ESR spectroscopy, enabling the characterization of its surrounding environment. Figures 1C and 1D show the ESR spectra of LMW TEMPOL and RNP, respectively. The ESR spectrum of LMW TEMPOL exhibited a triplet signal due to interactions between the 14N nuclei and the unpaired electron (Figure 1C). In contrast, RNP displayed a broad singlet signal, attributed to the confinement of nitroxide radicals within the core, leading to reduced mobility and interactions with adjacent nitroxide radicals within the polymer (Figure 1D). Thus, these ESR spectra provide crucial evidence that the PMNT segments constitute a solid core, while the PEG segments form an outer shell.3.2. Blood circulation of RNP after i.p. injectionAs discussed above, RNP has a core-shell structure of approximately 30 nm, covered by a PEG layer. Assessing its pharmacokinetics is essential. Since RNP contains TEMPO molecules, it can be detected via ESR spectroscopy, allowing in vivo monitoring. Due to the relevance of malaria parasite infection in erythrocytes, this study specifically examined its retention in the bloodstream after i.p. administration. Some of the nitroxide radicals in TEMPO are known to convert into ESR-inactive hydroxylamine forms in the body. Since hydroxylamine can also scavenge ROS, it was oxidized using potassium ferricyanide (K₃[Fe(CN)₆]) to regenerate its nitroxide form, enabling detection alongside the remaining nitroxide radicals. Figures 2A and 2B present the ESR spectra of plasma 1 h post-administration. The LMW TEMPOL exhibited sharp triplet signals, whereas RNP displayed a broad signal. This suggests that RNP, after being absorbed into the bloodstream from the peritoneal cavity, retained its particulate form even 1 h post-administration. Figure 2C illustrates the time profiles of ESR signals for RNP and TEMPOL in plasma following their i.p. injection. The ESR signal intensity of RNP in plasma was significantly higher than that of TEMPOL at 30 min post-injection. As shown in the inset, only 0.04% of the injected TEMPOL was detected at 30 min. In contrast, 11% of the injected RNP remained in the bloodstream after 1 h, and even after 24 h, 1% of the injected RNP was still circulating. The rapid clearance of TEMPOL is consistent with previous reports25 and is likely attributed to its rapid nonspecific diffusion throughout the body and subsequent renal elimination. Previous studies have reported that polymeric nanoparticles with high dispersion stability and biocompatibility are taken up by lymphatic vessels, reaching the bloodstream after i.p. administration.38 Once in circulation, RNP remains in the bloodstream for an extended period due to two advantages: the nanostructure prevents excretion through the kidney, and the PEG shell layer prevents protein adsorption.16 Although intracellular accumulation of RNP in infected erythrocytes was not directly assessed in this study, we have reported that RNP exhibits extremely low uptake into red blood cells16, suggesting that RNP exerts its antioxidant effects by scavenging extracellular ROS while circulating in the bloodstream.3.3. RNP treatment against the malaria model in miceFollowing intraperitoneal administration, RNP enters the bloodstream and remains detectable for over 24 h. Due to its prolonged circulation, we hypothesized that daily administration of RNP would effectively eliminate excess ROS in the blood and mitigate malaria-induced oxidative damage. To investigate whether RNP could ameliorate disease severity in the mouse malaria model by clearing ROS from the bloodstream, PBS, RNP, or TEMPOL was intraperitoneally injected daily from day 4 to day 11 post-infection with PbA (Figure 3A). All PbA-infected mice treated with PBS died by day 8 post-infection (Figure 3B). While TEMPOL treatment exhibited a trend toward improved survival, the difference was not statistically significant. In contrast, RNP treatment led to a significant improvement in survival compared to PBS (Figure 3B). Body temperature was measured as an indicator of disease severity.39, 40 Figure S1 shows individual changes in body temperature of treated mice until day 13. The body temperature of the uninfected group was approximately 36.6 ℃. In contrast, the body temperature of mice that died on days 7 and 8 dropped dramatically before death. Figure 3C summarizes the average body temperature of treated mice on day 6 using these data. On day 6, PBS-treated mice exhibited a significant drop in body temperature (34.4 ℃), indicating severe disease progression. TEMPOL-treated mice maintained slightly higher temperatures (35.8 ℃), though the difference was not statistically significant. Notably, RNP-treated mice maintained a body temperature (36.5 ℃) comparable to healthy controls, suggesting effective ROS scavenging and protection against systemic severe malaria-induced damage. Thus, RNP administration effectively suppressed fatal systemic damage in the mouse malaria model.  Given RNP’s prolonged retention in the bloodstream, we next examined its role in mitigating disease progression. First, we investigated the effect of RNP administration on parasite infection and proliferation. To confirm the presence of malaria parasites, blood was collected from the tail to prepare Giemsa-stained blood smears. Parasitemia was then evaluated through microscopic observation of these smears. Figure 4 illustrates individual changes in parasitemia (%) in PbA-infected mice in the PBS-, RNP-, and TEMPOL-treated groups between day 3 and day 13. Parasitemia in PbA-infected mice gradually increased over time. The parasitemia in the surviving TEMPOL- and RNP-treated mice reached approximately 30 to 40% on day 13 (Figure 4). Despite improving survival and disease severity, RNP did not reduce parasitemia, supporting the conclusion that its therapeutic effect is not due to antiparasitic activity. Instead, RNP scavenges extracellular ROS in the bloodstream, thereby suppressing malaria-induced oxidative damage and improving host resilience without interfering with parasite replication.3.4. Oxidative stress in the bloodIt has been reported that oxidative stress increases in the blood during malaria parasite infection.41 To assess ROS levels in the blood, superoxide anion was detected using DHE, which emits fluorescence upon reaction with superoxide anion. Measurements were taken on day 1, 4, and 6 after infection. The level of superoxide anion did not increase on days 1 and 4 (Figure S2). However, ROS levels sharply increased on day 6 after infection (Figure S2). Although parasite proliferation surged on day 8 (Figures 4B and 4C), ROS levels increased before this rapid parasite growth. From these results, PbA-infected mice treated with PBS as a positive control died before the surge in parasite levels, strongly suggesting that ROS played a significant role in this outcome. Here, we measured superoxide anion levels in the plasma of TEMPOL- and RNP-treated mice on day 6 post-infection (Figure 5A). TEMPOL treatment did not reduce superoxide anion levels in plasma, while RNP treatment significantly decreased these levels (P < 0.05). Thus, ROS levels increased sharply before the rapid proliferation of parasites, and RNP effectively suppressed this increase. To evaluate the impact of elevated ROS in the bloodstream, particularly on erythrocytes, we assessed their membrane oxidation by measuring lipid peroxidation in erythrocytes on day 6 (Figure 5B). On day 6 post malaria parasite infection, lipid oxidation in erythrocytes was significantly increased. Similar to its insufficient scavenging of ROS in the blood, TEMPOL treatment did not effectively prevent lipid oxidation in erythrocytes. In contrast, RNP significantly reduced lipid oxidation in erythrocytes, showing a statistically significant difference compared to the PbA-infected group (P < 0.05). Oxidation of erythrocyte membranes induces hemolysis, leading to the leakage of hemoglobin. Hemoglobin released from erythrocytes exacerbates oxidative stress in the host, contributing to the severity of life-threatening malaria-related complications.42, 43 Here, the effect of RNP treatment on hemolysis was evaluated using blood from PbA-infected mice on day 6 post-infection. The osmotic fragility of erythrocytes was measured as a function of hypotonic salt concentrations (Figure 6A). At the same NaCl concentrations, the extent of hemolysis in the PbA-infected groups with PBS treatment was significantly higher than that in the uninfected group with PBS administration (Figure 6A). Consistent with previous findings, TEMPOL treatment did not show any significant effects. In contrast, the extent of hemolysis in the RNP-treated group was markedly lower than that in the PbA-infected group with PBS administration (Figure 6B, P < 0.1). Since ROS cause severe tissue damage, their efficient removal from the bloodstream may improve survival rates and disease outcomes. The disconnect between unchanged parasitemia and improved clinical parameters highlights the importance of host-targeted antioxidant therapy, particularly in infections where inflammation and oxidative stress drive pathogenesis. Although TEMPOL showed a slight trend toward improved survival, this difference did not reach statistical significance under the tested conditions. While TEMPOL may confer transient antioxidant benefits during the early phase of infection, its rapid clearance and limited persistence in the bloodstream likely hindered its therapeutic potential. Moreover, its intracellular accumulation may disturb redox homeostasis and reduce overall efficacy, particularly during early-stage intervention.13 These characteristics of TEMPOL contrast with the sustained effect observed for RNP, reflecting their distinct pharmacokinetic and functional profiles. While our study focused on oxidative stress in the bloodstream, it is increasingly recognized that ROS produced in tissues such as the liver and spleen, as well as by immune cells including neutrophils and monocytes, also play significant roles in malaria pathophysiology. In this context, not only the antioxidant ability of RNP, but also the immunomodulatory potential of its PEG shell layer warrant further investigation. To fully elucidate the therapeutic mechanisms of RNP, future studies will focus on pharmacokinetics and intracellular distribution, using fluorescently labeled RNP and flow cytometry-based assays to assess uptake in both infected and uninfected erythrocytes. 4. ConclusionsIn this study, we investigated the effect of RNP treatment on infectious disease-induced oxidative stress in a mouse malaria model. Compared to the control group, RNP treatment significantly ameliorated disease severity and survival by effectively scavenging ROS in the bloodstream, preventing oxidative damage, and reducing the increased fragility of erythrocyte membranes caused by infection. These effects were attributed to the long-term circulation of RNP in the blood. Based on these findings, RNP treatment represents a promising therapeutic strategy for mitigating malaria severity. 5. Supporting InformationIndividual body temperature of the infected mice; levels of superoxide anion on day 1, 4, and 6 post infection in the plasma of PbA-infected C57BL/6J mice.6. AcknowledgmentsWe would like to thank Dr. Takuto Toriumi (University of Tsukuba, Japan) for his support in measuring TBARS in erythrocytes and assessing osmotic fragility. We also appreciate Dr. Pennapa Chonpathompikunlert (Thailand Institute of Scientific and Technological Research, Thailand) for her valuable discussions regarding the i.p. injection of RNP. Additionally, we thank Dr. Takashi Imai and Dr. Yuki S. Tateishi (National Institute of Infectious Diseases, Japan) for their helpful discussions and technical assistance with the malaria mouse model. 7. FundingThis study was financially supported by a Grant-in-Aid for Specially Promoted Research (19H05458) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (YN) and Japan Agency for Medical Research and Development (AMED) under the Emerging and Re-emerging infectious Disease Research Program (Interdisciplinary Research Area) (21wm0325040h0001) (YN, TY, TAN). This work was supported in part by a grant for Research on Emerging and Reemerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (24fk0108683j2402, 24fk0108681j0602 and 24fk0108680h0002 to TAN), a grant from the Japan Society for the Promotion of Science (JSPS) (KAKENHI 23KK0142 to TAN and 23K14514 to TAR) and a grant from Takeda Science Foundation (90003200) to TAN. Cooperative Research Grant (2021-joint-3, 2022-joint-3, 2023-joint-15 and 2024-joint-5) of National Research Center for Protozoan Diseases Obihiro University of Agriculture and Veterinary Medicine to TAR.8. Declaration of Competing Interest University of Tsukuba possesses patents on RNP and is licensed to CrestecBio.9. Author ContributionsToru Yoshitomi: Investigation, Methodology, Formal analysis, Validation, Visualization, Data curation, Funding acquisition, Writing – original draft, Writing – review & editing. Kyoko Hayashi: Investigation, Validation. Tamasa Araki: Methodology, Writing – review & editing. Takeshi Annoura: Conceptualization, Methodology, Funding acquisition, Writing – review & editing. Yukio Nagasaki: Conceptualization, Funding acquisition, Writing – review & editing, Supervision, Project administration.10. References1. Schofield, L.; Grau, G. E., Immunological processes in malaria pathogenesis. Nat Rev Immunol 2005, 5 (9), 722-35.2. Venkatesan, P., WHO world malaria report 2024. Lancet Microbe 2025, 101073.3. Trampuz, A.;  Jereb, M.;  Muzlovic, I.; Prabhu, R. M., Clinical review: Severe malaria. Crit Care 2003, 7 (4), 315-23.4. Van den Steen, P. E.;  Deroost, K.;  Deckers, J.;  Van Herck, E.;  Struyf, S.; Opdenakker, G., Pathogenesis of malaria-associated acute respiratory distress syndrome. Trends Parasitol 2013, 29 (7), 346-58.5. Batte, A.;  Berrens, Z.;  Murphy, K.;  Mufumba, I.;  Sarangam, M. L.;  Hawkes, M. T.; Conroy, A. 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Gwozdzinski, K.;  Pieniazek, A.; Gwozdzinski, L., Reactive Oxygen Species and Their Involvement in Red Blood Cell Damage in Chronic Kidney Disease. Oxid Med Cell Longev 2021, 2021.FiguresFigure 1. (A) Schematic illustration of self-assembling redox nanoparticle (RNP). (B) Size distribution of RNP (C) ESR spectrum of TEMPOL in PBS (D) ESR spectrum of RNP in PBS. Illustration of RNP was reprinted with permission from [21].Figure 2. ESR spectra and time course of circulating nitroxide radicals in the bloodstream after i.p. injection (A, B) The ESR spectra of (A) TEMPOL and (B) RNP in plasma at 1 h after injection. (C) The time profiles of the circulating RNP (red) and TEMPOL (blue) in plasma after i.p. injection, which was determined by ESR signal intensity of nitroxide radicals in plasma. Signal intensities after adding potassium ferricyanide to re-oxidize hydroxylamines to nitroxide radicals mean the total drug concentrations (nitroxide radicals + hydroxylamines). Data expressed as mean ± standard error of the mean (SEM) (n = 4). Statistical analyses were conducted as a Student’s t-test; *p < 0.05.Figure 3. Improvement in the severe malaria condition by using RNP. (A) Treatment procedure. (B) Survival rate of post PbA-infection from day 0 to day 13. (n = 5). The cumulative survival rates from each treatment group were compared by the Kaplan–Meier survival analysis.; *p < 0.05. (C) Body temperature on day 6 post infection (n = 5). Statistical analyses were conducted as one-way ANOVA; *p < 0.05.Figure 4. Percent parasitemia (percentage of erythrocytes infected with PbA) over the course of infection in C57BL/6J mice from day 3 to day 13. Individual parasitemia in (A) PBS-treated group (B) TEMPOL-treated group, and (C) RNP-treated group from day 3 to day 13. Individual color corresponds to the plot color in Figure S1 (n = 5). Statistical analyses were conducted as one-way ANOVA; *p < 0.05.Figure 5. Levels of (A) superoxide anion in plasma and (B) lipid peroxidation in the erythrocytes on day 6 post PbA-infection. (A) FL intensity of DHE, which can detect superoxide anion, in plasma on day 6 post PbA-infection. (B) TBARS in the erythrocytes, which indicates the level of lipid peroxidation, on day 6 post infection. Data expressed as mean ± standard error of the mean (SEM) (n= 5). Statistical analyses were conducted as one-way ANOVA; *p < 0.05.Figure 6. Osmotic fragility of the erythrocytes in the PbA-infected mice. (A) Hemolyzed fraction of the erythrocytes as a function of the NaCl concentration between 0.32% and 0.9% of NaCl (w/v%). (B) Half-hemolytic concentrations of the NaCl. Data expressed as mean ± standard error of the mean (SEM) (n= 5). Statistical analyses were conducted as one-way ANOVA; *p < 0.05.2image3.tiffimage4.tiffimage5.tiffimage6.tiffimage1.tiffimage2.tiff