Peroxidase-Mimicking Nanoassembly Mitigates Lipopolysaccharide

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‘Peroxidase’ mimicking Nano-assembly Mitigates LipopolysaccharideInduced Endotoxemia and Cognitive Damage in the Brain by Impeding Inflammatory Signaling In Macrophages Santhosh Kalash Rajendrakumar, Vishnu Revuri, Manikandan Samidurai, Adityanarayan Mohapatra, Jae Hyuk Lee, Palanivel Ganesan, Jihoon Jo, Yong-kyu Lee, and In-Kyu Park Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02785 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 24, 2018

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‘Peroxidase’ mimicking Nano-assembly Mitigates Lipopolysaccharide-Induced Endotoxemia and Cognitive Damage in the Brain by Impeding Inflammatory Signaling In Macrophages Rajendrakumar Santhosh Kalash1‡, Vishnu Revuri2‡, Manikandan Samidurai1, 3, Adityanarayan Mohapatra1, Jae Hyuk Lee4, Palanivel Ganesan5, Jihoon Jo3, Yong-Kyu Lee2*, In-Kyu Park1* 1

Department of Biomedical Science and BK21 PLUS Center for Creative Biomedical Scientists

at Chonnam National University, Chonnam National University Medical School, Gwangju 61469, Republic of Korea. 2

Department of Green Bioengineering, Korea National University of Transportation, Chungju

27469, Republic of Korea. 3

NeuroMedical Convergence Lab, Biomedical Research Institute, Chonnam National University

Hospital, Jebong-ro, Gwangju 501-757, Republic of Korea.

4

Department of Pathology, Chonnam National University Hwasun Hospital, Chonnam National

University Medical School, Hwasun 58128, Republic of Korea.

ACS Paragon Plus Environment

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Nanotechnology Research Center, Department of Biotechnology and Applied Life Science,

College of Bio-medical and Health Science, Konkuk University Glocal campus, Chungju 380701, Republic of Korea.


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ABSTRACT Oxidative stress during sepsis pathogenesis remains the most important factor creating imbalance and dysregulation in immune cell function, usually observed following initial infection. Hydrogen peroxide (H2O2), a potentially toxic reactive oxygen species (ROS), is excessively produced by pro-inflammatory immune cells during the initial phases of sepsis and plays a dominant role in regulating the pathways associated with systemic inflammatory immune activation. In the present study, we constructed a peroxide scavenger mannosylated-polymericalbumin-manganese dioxide (mSPAM) nano-assembly to catalyze the decomposition of H2O2 responsible for the hyper-activation of pro-inflammatory immune cells. In a detailed manner, we investigated the role of mSPAM nano-assembly in modulating the expression and secretion of pro-inflammatory markers elevated in bacterial lipopolysaccharide (LPS)-mediated endotoxemia during sepsis. Through a facile one-step solution phase approach, hydrophilic bovine serum albumin-reduced manganese dioxide (BM) nanoparticles were synthesized and subsequently self-assembled with cationic mannosylated disulfide cross-linked polyethylenimine (mSP) to formulate mSPAM nano-assembly. In particular, we observed that the highly stable mSPAM nano-assembly suppressed HIF1α expression by scavenging H2O2 in LPS-induced macrophage cells. Initial investigation revealed that significant reduction of free radical by the treatment of mSPAM nano-assembly has reduced the infiltration of neutrophils and other leukocytes in local endotoxemia animal model. Furthermore, therapeutic studies in a systemic endotoxemia model demonstrated that mSPAM treatment reduced TNFα and IL-6 inflammatory cytokines in serum, in turn circumventing organ damage done by the inflammatory macrophages. Interestingly, we also observed that the reduction of these inflammatory cytokines by mSPAM nano-assembly further prevented IBA-1 immuno-positive microglial cell activation in the brain and


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consequently improved the cognitive function of the animals. Altogether, the administration of mSPAM nano-assembly scavenged H2O2 and suppressed HIF1α expression in LPS-stimulated macrophages and thereby inhibited the progression of local and systemic inflammation as well as neuroinflammation in an LPS-induced endotoxemia model. This mSPAM nano-assembly system could serve as a potent anti-inflammatory agent, and we further anticipate its successful application in treating various inflammation-related diseases. Keywords: Endotoxemia, manganese dioxide, lipopolysaccharide, bovine serum albumin, inflammation.


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Sepsis is a life-threating condition that accounts for 150 million deaths every year, and it has been reported to be the second most common cause of mortality in non-coronary intensity care units.1 Pathogenesis of sepsis is associated with the hyper-activation of the innate immune system and the spontaneous secretion of systemic inflammatory molecules such as proinflammatory cytokines and secretory proteins, in response to the stress induced by microbial infections.2 Lipopolysaccharide (LPS), an endotoxin present on the cell wall of gram-negative bacilli, plays a dominant role in the microbe-induced sepsis. Binding of LPS to toll-like receptor 4 (TLR4) on the surface of the immune cells such as macrophages or other scavenging cells triggers several intracellular inflammatory pathways such as the nuclear factor-κB (NF-κB) pathway and promotes secretion of pro-inflammatory components such as hydrogen peroxide (H2O2), nitric oxide species (NOS), prostaglandin E2 (PGE2), cyclooxygenase (COX)-2, tumor necrosis factor α (TNF-α), IL-6 and IL-1β.3 The excessive secretion of pro-inflammatory cytokines can have deleterious effects in the organs, leading to tissue damage and multi-organ dysfunction and eventually resulting in patient death.4 Several studies reported that recurrent insults from inflammogens such as LPS result in microglial cell activation, leading to neurodegradation and the induction of numerous neurological diseases such as Alzheimer’s disease and Parkinson’s disease.5,6 Despite the severity of the complications associated with sepsis, effective therapeutic strategies in clinical settings have yet to be achieved. Although drotrecogin alpha has been approved by the FDA for the treatment of severe sepsis, few issues associated with this compound, including high costs, low efficacy and poor biological safety, limited its usage in clinical settings.7 Recent findings suggested that prolonged oxidative stress and damage associated with sepsis could aggravate disease progression.8 In the course of sepsis, upregulated systemic inflammation


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results in hyper-metabolic activation of innate immune cells, causing deterioration of endogenous antioxidative buffering capacity and subsequent secretion of reactive oxygen species (ROS).9 Hydrogen peroxide (H2O2), an extremely toxic ROS, is usually over-produced in scavenger cells during sepsis.10 The activation of NADPH oxidases by LPS generates superoxide products, culminating in the production of H2O2 in inflammatory cells.11,12 The excessive secretion of hydrogen peroxide results in the activation of inflammatory cytokines and markers.13 It also serves as a neutrophilic chemo-attractant, which induces non-specific neutrophil infiltration and organ damage.10 Moreover, H2O2 accelerates the release of the secondary proinflammatory molecule such as HMGB1 from activated macrophages and thus intensify the inflammation caused by activating immune cells in other major organs.14 Hypoxia inducing factor 1α (HIF1α) has an essential role in the development of LPS-induced sepsis as it promotes the secretion of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-4, IL-6 and IL-12.15 During inflammation, elevated intrinsic H2O2 promotes gene expression of HIF1α by activating its

transcriptional

macrophages.16,17

promoter, In

addition,

Ca2+/calmodulin-dependent

thereby HIF1α

protein

indirectly

activating

activation

kinase

II

is

the

regulated

(CAMKII)

in

NF-κB by

pathway

in

H2O2-dependent

inflammatory

cells.18

Organoselenium-based antioxidant compounds are currently used to reduce systemic insults incurred from oxidative damage during sepsis.9 However, discouraging results from the clinical trials indicated no significant reduction in pro-inflammatory cytokine secretion and poor efficacy in improving mortality rates with selenium-based therapies.19 These findings necessitate the development of a novel and potent antioxidant-based therapy to control H2O2 levels in inflammatory immune cells for alleviating the chain of inflammatory actions during sepsis.


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In the present study, we developed a mannosylated disulfide cross-linked polyethylenimine (ssPEI) (mSP)-coated bovine serum albumin-reduced MnO2 (mSPAM) nano-assembly as a potent H2O2 scavenger and investigated their anti-inflammatory activity in LPS-induced systemic and local inflammation murine models (Figure 1). Mannose was conjugated to the nanoassembly to effectively target immune cells20,21. Although, various novel polymers and metalbased nanoparticles are used for scavenging H2O2,22–24 manganese oxide (MnO2) are the most predominant nanomaterials, that can specifically decompose toxic hydrogen peroxide (H2O2) to oxygen and water.25 Here, through a facile one-step approach, we developed a stable watersoluble BSA/MnO2 nanoparticle (BM; Figure S2a) by chemically reducing KMnO4 using albumin as the template.26 A detailed description on the synthesis of polymeric mSP, BM and mSPAM nano-assembly is provided in the supporting text (Figure S1-S9). The formulation of mSPAM nano-assembly was mediated by electrostatic spontaneous condensation between negatively charged BM and positively charged mSP. The spherical morphologies of the nanomaterials are shown in Figures 2a and 2b. From TEM images (Figure 2a) and EDS spectra, it can be inferred that BM attained spherical morphology, where the coronal surface of BSA was covered with MnO2 (Figure S3a). TEM images shown in Figure 2b also confirmed the spherical morphology of the mSPAM nano-assembly with average size of 97 nm and surface charge of +17.8 ±3 mV (Figure 2c) at the weight ratio of 1:2 w/w (BM:mSP). XPS analysis was performed to investigate the elemental mappings of the BM and mSPAM nano-assembly (Figure S5 and S6). As shown in Figure 2d and S6, we confirmed the presence of manganese atom along with carbon, oxygen and nitrogen atoms in mSPAM. The higher-resolution manganese spectra in Figure 2e also displayed the presence of Mn 2p1/2 and Mn 2p3/2 mSPAM nano-assembly. Moreover, the spin-orbital splitting distance between the Mn 2p1/2 and Mn 2p3/2 peaks, approximately 11.7 eV,


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signified the presence of MnO2 in mSPAM nano-assembly.25 FT-IR spectra further confirmed the presence of MnO2 in the synthesized mSPAM. In Figure 2e, the peak at 540 cm-1 showed the presence of Mn in the BM nanoparticle, and the peak shift near 1400 cm-1 indicated the strong interaction between the carboxyl COO- groups of BSA and the manganese ions.27 Interestingly, BSA template has greatly improved the stability of MnO2 (Figure 2f). Moreover, the coating of mSP over BM nanoparticle has shown negligible effects over the stability of the mSPAM nanoassembly (Figure 2f). These characterizations showed that the synthesized nano-assemblies were stable in biological environments. H2O2 triggers the generation of free radicals responsible for the initiation of immune activation.10 Therefore, we used terephthalic acid (TA) as a H2O2 -sensing fluorescent probe to investigate the ability of mSPAM nano-assembly to reduce H2O2-mediated free radical production.28 As shown in Figure 2g, the fluorescence of TA was significantly increased in the presence of H2O2. However, after the addition of mSPAM nano-assembly, the fluorescence of TA in the presence of H2O2 was significantly reduced. Moreover, the fluorescence intensity of TA was further reduced with increasing concentration of mSPAM nano-assembly. Because mSPAM catalytically reduced H2O2, the formation of free radicals from H2O2 was clearly inhibited. This result indicated that mSPAM nano-assembly can effectively quench the production of H2O2-mediated free radicals. Moreover, mSPAM nano-assembly were selectively towards H2O2, but not towards other superoxide free radicles (Figure S10). Before investigating the biomedical applications of mSPAM, it was important to investigate the toxicology profiles of the nano-assembly. As shown in Figure 2h, no significant reduction in cell viability was observed in mSPAM-treated RAW264.7 macrophage cells at 24 h post-


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incubation. Therefore, we believed that mSPAM nano-assembly was non-toxic and safe for in vitro and in vivo investigations. Schematics in Figure 3a represent the mechanism of anti-inflammation by mSPAM nanoassembly in LPS-treated RAW264.7 macrophages. For effective therapeutic activity, the nanomaterials should efficiently enter the cells and generate the desired scavenging effect without noticeable toxicity. Hence, the intracellular uptake of mSPAM nano-assembly in the RAW264.7 cell line was investigated. The BM nanoparticle was conjugated with FITC fluorescent dye to visualize the cellular internalization of mSPAM nano-assembly. As shown in Figures 3b and S11, the enhanced fluorescent intensity of FITC-mSPAM nano-assembly was clearly noticed in RAW264.7 cells compared to FITC-BM nanoparticle-treated cells. Interestingly, the intracellular accumulation of FITC-mSPAM nano-assembly in LPS-treated RAW264.7 cells was markedly higher than in non-treated RAW264.7 cells. This clearly emphasized that coating of mSP over BM nanoparticle has enhanced intracellular uptake in LPSstimulated RAW264.7 cells. To further, confirm the intracellular uptake mechanism of mSPAM in LPS-stimulated RAW264.7 cells, endocytic inhibitors such as chlorpromazine,29 monodansylcadvarine,30 and methyl-β-cyclodextrin31 that inhibits clathrin-, receptor- caveolaemediated endocytosis were employed. For blocking mannose receptor and macropinocytosis, we have used mannan32 and amiloride33. In figure S12a, based on flow cytometry analysis of LPSstimulated RAW264.7 cells incubated with mSPAM nanoassembly in the presence of each endocytic inhibitor, the intracellular localization of mSPAM nanoassembly in LPS-stimulated RAW264.7 cells was significantly (approximately 50%) reduced after co-incubation with mannan, methyl-β-cyclodextrin, and monodansylcadvarine. Similar results were observed in Figure S12b, where the confocal images demonstrated that the intracellular localization of


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mSPAM (represented by green fluorescence of FITC-mSPAM nanoassembly) was greatly diminished in presence of methyl-β-cyclodextrin, mannan, and monodansylcadvarine. These in vitro uptake studies indicate that intracellular uptake of mSPAM nanoassembly by the LPSstimulated RAW264.7 cell line was facilitated through mannose receptor-mediated endocytosis. Additionally, the intracellular uptake of mSPAM nanoassembly involved calveolae-mediated endocytotic pathway, which can be attributed towards the presence of polyethylenimine in mSPAM formulation. Previous studies have reported that the uptake of polyethylenimine based nanoparticles in the cells were dominated by the caveolae-mediated endocytosis.37,38 Further investigation also revealed that either LPS pretreatment or co-treatment with mSPAM showed similar elevated intracellular uptake in RAW264 with respect to increase in time (figure S13a, b). Based on these studies, we confirm the intracellular uptake of mSPAM nanoassembly in LPSstimulated RAW264.7 cells through mannose receptor and caveolae mediated endocytosis. Endotoxins induce secretion of ROS through the TLR-4-mediated NADPH oxidase pathway.13 Elevated ROS levels mediated by the LPS/TLR4 signaling cascade in macrophages triggered the immune response through production of H2O2 by superoxide dismutase.39 Therefore, the efficacy of mSPAM nano-assembly in reducing the levels of ROS and H2O2 production in macrophage cells was investigated. As shown in Figure 3c and Figure S14, macrophages treated with mSPAM nano-assembly displayed less production of intracellular H2O2 than did LPS-treated and BM/LPS-treated groups. Later, we studied the effects of mSPAM in reducing the levels of ROS and H2O2 production in RAW 264.7 cells. The results presented in Figures 3d and S15 further demonstrate that the LPS treatment of macrophages stimulated ROS production. Interestingly, the mSPAM treated cell lines showed reduced ROS production compared to non-targeted SPAM or BM.


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Inducible nitric oxide synthase (iNOS) expressed from LPS-stimulated macrophages can accelerate the secretion of the inflammatory mediator nitric oxide (NO).2,40 Therefore, further investigation of NO production in LPS-induced cells treated with mSPAM nano-assembly was performed (Figure 3e). Levels of NO were greatly reduced in macrophages treated with mSPAM nano-assembly and were comparable to those of negative control groups (cells without LPS treatment). However, the data presented in figure S16, shows that BM, SPAM or mSPAM alone (without LPS treatment) did not induce production of inflammatory markers such as ROS, H2O2 and NO in RAW264.7 cell line, confirming mSPAM to be a safe and nontoxic nanoassembly in the biological system. Then, expression patterns of P-IKK and pP-65 proteins responsible for the activation of TLR-4mediated NF-κB pathway in immune cells were studied. As shown in Figures 3f and S17, cells treated with mSPAM nano-assembly showed significant reduction in expression of pP-65 proteins. Moreover, expression of pro-inflammatory markers iNOS and COX-2 were highly suppressed after treating LPS-stimulated RAW 264.7 cells with mSPAM nano-assembly, although there was no significant change in p-IKK expression level in mSPAM nano-assemblytreated RAW264.7 cells compared with the LPS control. Interestingly, HIF1α protein expression was significantly reduced by mSPAM nano-assembly compared to the LPS control group. It has been reported that HIF1α activation induced expression of pP-65.41–43 Based on these mechanistic studies, we concluded that mSPAM nano-assembly did not stimulate the NF-κB/pIKK pathway, whereas it inhibited pP-65 expression through suppressing HIF1α expression by scavenging H2O2. After confirming successful reduction in pro-inflammatory activity by mSPAM nano-assembly in vitro, the therapeutic role of mSPAM nano-assembly in preventing LPS-induced H2O2


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production in tissue-residing macrophages was investigated in a local inflammation mouse model (Figure 4a). L012 luminol agent shows luminescence only in the presence of ROS produced from H2O2 in LPS-injected tissues. Therefore, intraplantar injection of LPS was performed in both paws, and the mSPAM nano-assembly was subsequently injected in the same site after 4 h. The ROS levels in the paw site were visualized using L012 luminol dye at 4 h postsample injection. As shown in Figure 4b, mSPAM nano-assembly treatment reduced LPSinduced inflammation and neutrophil recruitment in mouse paws. As seen in Figure 4b and 4c, LPS injection in mouse paws showed higher L012 luminescent signals, whereas the signal was drastically lower in the mSPAM nano-assembly treatment group than in the BM- and SPAMtreated groups. This result attributed to the successful catalysis of H2O2 secreted during inflammation by mSPAM nano-assembly and regulation of neutrophil infiltration in the paw (Figure S18). Ruiz-Miyazawa et al. reported that vinpocetine reduced oxidative stress signals such as H2O2 during LPS-induced inflammation in paw tissue, confirming that neutrophil infiltration at the inflammation site was prevented.44 Even though mSPAM nano-assembly prevented infiltration of neutrophils and leukocytes to LPS-induced inflammation sites, its role as a H2O2 scavenger in prevention of immune cell infiltration is yet to be studied in a more detailed manner. During sepsis, LPS released from gram-negative bacteria escalates the secretion of proinflammatory cytokines such as TNFα and IL-6 in the circulation. Hence, the effect of mSPAM nano-assembly in suppression of pro-inflammatory cytokines and inflammatory markers in LPSinduced systemic inflammation model was investigated. C57BL/6 mice were injected with mSPAM nano-assembly (25 mg/kg) along with LPS (1 mg/kg) by i.p. injection. The levels of TNF-α and IL-6 were measured at 0, 2, 4 and 6 h after LPS injection. LPS administration


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induced elevated production of TNF-α and IL-6 at 2 h and 4 h post-injection, as shown in Figure 4d and 4e, respectively. The co-administration of LPS along with mSPAM nano-assembly inhibited TNF-α production significantly at 2 h and 4 h compared to the LPS only-treated group, whereas IL-6 production was reduced up to 73% and 72% at 2 h and 4 h post-injection, respectively. As seen in Figure 4f, histopathology of liver, lung and kidney tissues isolated from 24 h treatment groups were analyzed using H&E staining. In LPS-treated tissues, lung histology showed thickening of alveolar septa, pulmonary edema, neutrophil infiltration and hemorrhage.44 Liver histology displayed infiltration of inflammatory cells into the cavities of liver tissues and vacuolar degeneration in LPS-treated mice. A loss of renal tubular epithelial cells and decrease in brush borders and renal epithelial cells were clearly noticed on kidney histology (Figure 4f).45,46 All these abnormalities were found to be absent in the mSPAM nano-assembly treatment group. Hence, further investigation was carried out to analyze the expression of proinflammatory genes such as IL-1β, HMGB1, HIF1α and COX2 in major organs such as liver, kidney and lung isolated from treated mice using quantitative real-time polymerase chain reaction (qRT-PCR). mSPAM nano-assembly treatment showed significant reduction in IL-1β, HIF1α and COX2 transgene expression in liver (Figure S19a), lung (Figure S19b) and kidney (Figure S19c) compared with the LPS-treated group. However, the organs of the SPAM- or BMtreated animals did not show significant changes in terms of gene expression compared to LPStreated animals. Secretory HMGB1 proteins are lethal mediators of LPS-induced systemic inflammation and are released by activated macrophages, further orchestrating the secretion of other pro-inflammatory cytokines such as TNF-α and IL-6.47 Following LPS induction in mouse models, treatment with mSPAM nano-assembly produced significant reduction in gene expression of HMGB1 in the liver, lung and kidneys compared to BM- and SPAM-treated


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animals. IL-10, an anti-inflammatory cytokine, regulates the anti-inflammatory response by selectively inhibiting NF-κB gene transcription.48 Interestingly, transgene expression levels of IL-10 in the liver and kidney were similar in all treatment groups, whereas there was slight elevation of IL-10 gene expression in lung tissue of the SPAM and mSPAM nano-assemblytreated group (Figure S20). Overall, mSPAM nano-assembly treatment inhibited expression of pro-inflammatory proteins in the major organs and therefore suppressed secretion of proinflammatory cytokines such as TNF-α and IL-6 in the serum. Based on our previous study, we inferred that LPS administration increased blood levels of pro-inflammatory cytokines such as TNF α and IL-6. Studies showed that elevated plasma levels of inflammatory cytokines such TNF-α and IL-6 trigger immune activation in brain microglial cells, resulting in induction of neuro-inflammatory disorders such as Alzheimer’s or Parkinson’s disease (Figure 5a).49–51 Furthermore, pro-inflammatory mediators such as NOS, PGE2 and COX-2 are also activated by IL-6 and TNF-α in microglial cells.52–54 Immunoblot analysis showed enhanced expression of NF-κB, COX-2 and iNOS in mice treated with LPS (Figure 5b). Interestingly, the levels of these pro-inflammatory markers P-IKK, P-P65, P-P65 and P-IKK, were significantly reduced in SPAM-treated mice. The enhanced expression of inflammatory mediators alters synaptic plasticity and inhibits long-term potentiation.55,56 To evaluate the therapeutic efficiency of mSPAM nano-assembly in an LPS-induced neuroinflammation model, long-term potentiation (LTP) analysis through electrophysiological field recording was employed. The administration of LPS significantly impaired LTP (118.4 ± 3) compared with control (159.6 ± 5). The co-administration of mSPAM nano-assembly with LPS significantly prevented or reversed LP-induced LTP impairment (149.3 ± 3); however, such significance was not observed with BM or SPAM co-treatment (Figure 5c). Reactive microglia activate the major


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transcription factor nuclear factor κB (NF-κB) pathway

49,50

and enhance expression of pro-

inflammatory cytokines COX-2 and iNOS, leading to neuroinflammation.50,52 IBA-1 immunostaining was performed to confirm whether systemic administration of mSPAM nanoassembly reduced activation of microglial cells. Figure 5d shows that administration of mSPAM nano-assembly effectively reduced activation of microglia to levels similar to that of the control group (mice without LPS treatment). Taken together, these data indicate that mSPAM nanoassembly attenuated LPS-induced neuroinflammation and promoted brain homeostasis. In humans, sepsis is a critically dangerous condition caused by infectious bacteria continuously releasing endotoxins into the blood. LPS-induced endotoxemia is one of the crucial factors responsible for the pathogenesis of sepsis.57–59 H2O2, a toxic ROS, plays a dominant role in LPS/TLR4-mediated activation of the pro-inflammatory NF-κB signaling60,61 cascade via HIF1α62–65 and simulates secretion of various pro-inflammatory markers such as TNFα,66,67 IL-6, COX-2, HMGB1 and IL-1β,68 which mediate the pathophysiology of sepsis.69 We hypothesized that minimizing the levels of H2O2 in pro-inflammatory cells would have beneficial effects on the regulation of LPS-induced endotoxemia. Therefore, this study aimed to develop an H2O2 scavenger nano-assembly that could regulate secretion of LPS induced inflammatory markers and cytokines expressed by macrophages. Here, we used a manganese oxide-based scavenger nanoparticle, mSPAM nano-assembly, to mimic peroxidase-like activity and reduce levels of a toxic oxidizing agent, H2O2, in LPS-induced sepsis animal models. Conventional metallic nanomaterials produce unwanted long-term accumulation and retention in organ systems after treatment, resulting in unnecessary immune activation, whereas MnO2 nanomaterials are catalytically degraded to Mn2+ ions, with reduced retention time in the body and minimal exposure to innate immune cells.70–72 Moreover, the use of serum albumins for the fabrication of


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MnO2 nanoparticles has been beneficial in terms of high stability and low cytotoxicity, leading to improved biocompatibility.73 mSPAM nano-assembly formulation was based on electrostatic interaction between the cationic polymer and anionic protein-metal hybrid that can be highly suitable in clinical settings, since they prevent the release of toxic by-products that are generated from covalently conjugated polymer degradation.74,75 In the current study on intracellular mechanisms, we found that mSPAM nano-assembly showed enhanced suppression of HIF-1α-mediated NFκ-B activation by reducing intracellular levels of H2O2. Collective results from local, systemic and neuroinflammatory studies demonstrated that intraperitoneal administration of mSPAM nano-assembly effectively reduced the production of pro-inflammatory markers and resulted in alleviating LPS-induced endotoxemia. These results suggested that reduction of H2O2 by mSPAM nano-assembly gave rise to suppression of HIF1α mediated NF-κB activation, further preventing expression of pro-inflammatory cytokines. Apart from anti-inflammatory agent treatment and extracorporeal therapy, treatment strategies are quite limited for LPS-induced endotoxemia.76–78 MnO2-based nanomaterials have been widely studied due to their H2O2-scavenging properties for mitigating hypoxic levels in tumors, whereas their efficiency in regulation of LPS-induced sepsis and immune activation has not yet been studied until now.79,80 We harnessed the H2O2 -scavenging efficacy of mSPAM nano-assembly to mitigate H2O2 levels in LPS-induced sepsis models. Abnormal elevation of pro-inflammatory cytokines during LPS-induced endotoxemia has indirect effects on cognitive impairment. In short, pro-inflammatory cytokines activate microglial cells, in turn causing damage to neural cells in the brain. In the present study, we showed that LPS administration activated Iba1+ microglial cells in mouse brain. The electrophysiological characterization of excised brain slices further demonstrated impairment in


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cognitive function after LPS treatment in mice. The loss of cognitive function in LPS-treated mice can be attributed to the activation of microglial cells in the brain due to elevated levels of TNFα and IL-6 in the blood. Interestingly, suppression of TNFα and IL-6 secretion after the coadministration of LPS and mSPAM nano-assembly prevented the activation of Iba1+ microglial cells and improved the cognitive function in mice. In conclusion, our study detailed the distinctive H2O2 scavenging properties and efficient antiinflammatory activities of mSPAM nano-assembly to treat LPS-induced endotoxemia. This is the first study demonstrating the effect of H2O2 scavenging in alleviation of LPS-induced endotoxemia and neuro-inflammation. Here, new insights have also been identified regarding the anti-inflammatory mechanisms and treatments with manganese-based nanomaterials. Further exploration of these nanomaterials with other synergistic approaches hold great promise for inflammation research. In the future, the design of hybrid hydrogen peroxide nanoscavengers such as mSPAM nano-assembly could support the treatment of endotoxemia-mediated inflammation in sepsis. Corresponding Author * In-Kyu Park Email: [email protected] Department of Biomedical Science and BK21 PLUS Center for Creative Biomedical Scientists at Chonnam National University, Chonnam National University Medical School, Gwangju 61469, Republic of Korea * Yong-Kyu Lee Email: [email protected]


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Department of Green Bioengineering, Korea National University of Transportation, Chungju 27469, Republic of Korea Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENT This

work

was

financially

supported

by

Basic

Science

Research

Program

(No.

2016R1A2B4011184) and the Bio & Medical Technology Development Program (NRF2017M3A9E2056374) through the National Research Foundation of Korea (NRF) funded by the Korean government, MSIP. This work was also financially supported by The Leading Human Resource Training Program of Regional Neo industry through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2016H1D5A1910188) and NRF2015R1D1A1A09060567 National Research Foundation of Korea (NRF). Supporting Information Available: experimental methods; Chemical synthesis and characterization; additional data; additional figures, and works cited.


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Figure Legends Figure 1: Schematic representation on formulation of mSPAM nano-assembly and its role in inducing anti-inflammation in LPS induced sepsis mouse model. mSPAM was synthesized by facile one-step electrostatic interaction approach using BSA-MnO2 (BM) nanoparticle complexed with cationic ssPEI-mannose polymer (Top section). LPS induce H2O2 production in macrophages cells, thereby causing secretion of proinflammatory cytokines like TNFα and IL-6. The secreted proinflammatory cytokines activate microglial cells, leading to neural cell death and neuroinflammation (bottom left section). mSPAM suppression of H2O2 in macrophages cells has led to prevention of proinflammatory cytokines secretion and thus providing neuroprotection indirectly (bottom right section). Figure 2: Physiochemical characterization of mSPAM nano-assembly and its toxicity profile in RAW264.7 cell line. a) TEM image of BM, b) TEM image of mSPAM nano-assembly, c) hydrodynamic size and zeta potential of mSPAM nano-assembly formulation at different weight ratio (mSP:BM), d) and e) XPS of mSPAM nano-assembly, f) FTIR of BM, g) H2O2 scavenging assay using Terepthalic acid, and h) cell viability of RAW 264.7 cell line treated with mSPAM nano-assembly, . Figure 3: Mechanism of mSPAM nano-assembly reduce inflammatory marker via hydrogen peroxide scavenging in RAW264.7 cell line. a) schematic representation of mSPAM nano-


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assembly alleviating LPS induced inflammatory protein expression in RAW264.7 cell line, b) intracellular uptake of mSPAM nano-assembly in RAW264.7 cells treated with and without LPS, c) intracellular H2O2 level in mSPAM nano-assembly treated LPS stimulated RAW264.7 cell line using PO assay, d) ROS level in mSPAM nano-assembly treated LPS stimulated RAW264.7 cells using DCFDA assay, e) Nitrite level in mSPAM nano-assembly treated LPS stimulated RAW264.7 cells, and f) western blot analysis of proteins in NF-κB pathway in RAW264.7 treated with mSPAM nano-assembly. The concentration of LPS and mSPAM used was 1µg/ml and 100µg/ml (1:2 w/w – mSP:BM) respectively. (n = 4, SEM, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001) Figure 4: mSPAM nano-assembly alleviation of LPS induced local and systemic inflammation in C57BL/6 mice. a) schematic representation of local and systemic inflammation induction by LPS in C57BL/6 mice, local inflammation analysis in mice paw injected with LPS (1mg/kg) and mSPAM nano-assembly (25mg/kg) using L012 luminol, b) luminescence image of the mSPAM nano-assembly treated mice, and c) plot of relative level of inflammation measured based on the luminescent intensity in the paw region, ELISA analysis of d) TNFα and e) IL-6 level in serum at different time point, and f) Histology of live, lung and kidney from LPS induced mice treated with mSPAM nano-assembly. (n = 6, SEM, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001) Figure 5: Cognitive function and anti-inflammation in mSPAM nano-assembly treated LPS induced C57BL/6 mice. a) Schematic representation of mSPAM nano-assembly (25mg/kg) indirect role in alleviating neuroinflammation in LPS (1mg/kg) treated mice, b) western blot analysis of inflammatory marker expression in brain tissue of mSPAM nano-assembly treated LPS induced mice, c) electrophysiology analysis of mSPAM nano-assembly treated LPS induced mice brain, d) IBA-1 fluorescence staining of mice brain.


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Figures

Figure 1.


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Table of content:


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mSPAM nano-assembly scavenged H2O2, suppressed HIF-1α expression in LPS-stimulated macrophages and significantly inhibited the progression of local, systemic and neuroinflammation in LPS induced endotoxemia model. 80x39mm (300 x 300 DPI)


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Peroxidase-Mimicking Nanoassembly Mitigates Lipopolysaccharide

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