Impact of Morphology on Iron Oxide Nanoparticles-Induced

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Biological and Medical Applications of Materials and Interfaces

Impact of Morphology on Iron Oxide NanoparticlesInduced Inflammasome Activation in Macrophages Liu Liu, Rui Sha, Lijiao Yang, Xiaomin Zhao, Yangyang Zhu, Jinhao Gao, Yunjiao Zhang, and Longping Wen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17474 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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ACS Applied Materials & Interfaces

Impact of Morphology on Iron Oxide Nanoparticles-Induced Inflammasome Activation in Macrophages

Liu Liu1 #, Rui Sha1 #, Lijiao Yang4, Xiaomin Zhao2,3, Yangyang Zhu2,3, Jinhao Gao4*, Yunjiao Zhang2,3* and Long-Ping Wen 1*

1School

of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China 2Department of Colorectal & Anal Surgery, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 510180，China 3Nanobio Laboratory, Institute of Life Sciences, South China University of Technology, Guangzhou, Guangdong, 510006, China 4State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and iChEM, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China

#：These authors contributed equally to this study.

KEYWORDS: Iron oxide nanoparticles (IONPs), morphology, inflammasome,

pyroptosis, NLRP3, ROS generation, lysosomal damage, K+ efflux

ACS Paragon Plus Environment

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ABSTRACT Inflammasomes, a critical component of the innate immune system, mediate much of the inflammatory response manifested by engineered nanomaterials. Iron oxide nanoparticles (IONPs), a type of nanoparticles that have gained widespread acceptance in preclinical and clinical settings, are known to induce inflammasome activation, but how morphology affects the inflammasome-activating property of IONPs has not been addressed. In this report we have synthesized four morphologically-distinct IONPs having the same aspect ratio and similar surface charge, thus offering an ideal system to assess the impact of morphology on nanoparticle-elicited biological effect. We show that morphology was a critical determinant for IONPs-induced IL-1β release and pyroptosis, with the octapod and plate IONPs exhibiting significantly higher activity than the cube and sphere IONPs. The inflammasome-activating capacity of different IONPs correlated with their respective ability to elicit intracellular ROS generation, lysosomal damage and potassium efflux, three well-known mechanisms for nanoparticle-facilitated inflammasome activation. Furthermore, we demonstrate that the release of IL-1β induced by IONPs was only partly mediated by NLRP3, suggesting that inflammasomes

other

than

NLRP3

are

also

involved

in

IONPs-induced

inflammasome activation. Our results may have implications for designing safer nanoparticles for in vivo applications.

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INTRODUCTION Inflammation, our body’s first protective response upon challenge by ‘‘danger signals’’ coming from either exogenous or endogenous sources, constitutes an important part of nanomaterial-induced toxicity.1-3 Mounting work in the recent years has identified inflammasomes, a critical component of the innate immune system, as a major mediator of the inflammatory response manifested by engineered nanomaterials.4-5 Among the various inflammasomes, the NLRP3 inflammasome is the best studied.6-8 NLRP3 inflammasome activation is initiated by the assembly of the NLRP3 protein complex, which recruits and activates pro-inflammatory caspase 1. The active caspase 1 in turn promotes the cleavage of pro-IL-1β, resulting in the secretion of mature and functional IL-1β.9-10 NLRP3 inflammasome activation also leads to a pro-inflammatory form of programmed cell death known as pyroptosis.11-12 Excessive activation of inflammasome has been implicated in various diseases, such as Alzheimer’s disease, gout, atherosclerosis, and type-2 diabetes, necessitating the need for controlling the inflammasome activation.1, 13-16

To date, a large number of

engineered nanoparticles, including carbon, metal, metal oxide and polymeric nanoparticles, are known to activate NLRP3 inflammasome.15,

17-21

Among the

various mechanisms revealed, free radical (ROS) generation, lysosomal damage and potassium efflux have been shown to play key roles during nanoparticle-induced inflammasome activation.18, 22-24 However, our current understanding on what defines a nanoparticle’s ability to activate inflammasomes is still rather limited.

The biological effects of nanoparticles, oftentimes remarkable and sometimes unexpected, are largely dictated by their unique physicochemical properties for example, chemical composition, shape, size and surface properties. Among these 3



parameters,

particle

morphology

contributes

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significantly

to

the

inflammation-modulating property of nanoparticles. Shiuli et al. demonstrated that hydroxyapatite nanoparticle with fiber and dot morphology caused higher acute inflammation in vivo as compared to the nanoparticle with sheet morphology.25 Ji et al. suggested that CeO2 nanorods/nanowires exhibiting shorter lengths and lower aspect ratios may induce inflammasome activation.26 Oh et al. revealed that different shapes

of poly (3, 4-ethylenedioxythiophene) nanomaterials had different effect on

ROS production and proinflammatory response, with an inverse correlation observed between the aspect ratio and inflammation-related toxicity.27 Notably, in all of these studies, the nanoparticles being assessed, in addition to having different shape, also exhibited a difference in size, thus the differing inflammasome-modulating effects could not be solely attributed to the morphology.

Iron oxide nanoparticles (IONPs) are one type of the nanoparticles that have gained widespread acceptance in preclinical and clinical settings, notably as contrast agents and drug carriers.28-30 Recently their applications have expanded into areas such as treatment of iron deficiency31 and cancer immunotherapy.32 It is thus of no surprise that much research has been directed towards understanding the toxicological aspects of IONPs. A significant number of reports have clearly documented the inflammation-modulating

effects,

with

several

of

them

demonstrating

inflammasome-activating capability, for IONPs.33-36 However, none of the reports have addressed the issue of morphological impact on IONPs-induced inflammasome activation. Moreover, whether the observed inflammasome activation was mediated by NLRP3 has not been clearly documented.

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In this work we have synthesized four types of IONPs with different morphologies, namely octapod, plate, cube and sphere, respectively. These IONPs had nearly the same aspect ratio and similar surface charge, thus giving us an excellent system to evaluate the impact of particle morphology on the capacity of IONPs to activate inflammasomes. We revealed that morphology indeed matters, as significant difference in inflammasome-activation activity was observed for these nanoparticles, with

the

octapod

and

plate

IONPs

exhibiting

significantly

higher

inflammasome-activating activity than the cube and sphere IONPs. We also showed that the inflammasome-activating capacity of different IONPs correlated with their respective ability to elicit intracellular ROS generation, lysosomal damage and potassium efflux, three known mechanisms for nanoparticle-facilitated inflammasome activation. Finally, we demonstrated that the inflammasome activation induced by IONPs was only partially mediated by NLRP3, suggesting that inflammasomes other than NLRP3 also played an important role in IONPs-induced IL-1β release and pyroptosis.

EXPERIMENTAL SECTION Reagents. Nigericin, PMA, cytochalasin B, cytochalasin D, Mito TEMPO, VAS 2870 and silicon dioxide were from Sigma Aldrich. CCK8 cell viability kit was from Biosharp. LPS, Lysotracker and Lysosensor were from Invitrogen. Hoechst 33342, propidium iodide and 2,7-dichlorouorescein diacetate were from Beyotime. CA-074-Me was from Calbiochem. FAM-FLICA Caspase-1 Assays kit were from ImmunoChemistry Technologies. Anti-NLRP3 and anti-mouse caspase-1 (p20) antibodies were from Adipogen. Anti-mouse IL-1β antibody was from R&D. 5



Anti-human cleaved IL-1β was from Sangon Biotech. Anti-ASC was from Santa Cruz. Anti-GAPDH was from Millipore. Anti-human caspase-1, GSDMD and HMGB1 antibodies were from Cell Signaling Technology. Mouse IL-1β and human IL-1β ELISA kits were from R&D. Mouse TNF-α ELISA kit was from CUSABIO.

Synthesis of iron oxide nanoparticles (IONPs) with different morphology. We used a modified facile method to prepare IONPs with different morphology. Briefly, synthesized iron oleate (1 mmol) and of oleic acid (0.5 mmol) were mixed in 1-octadecene. The mixture was heated to different temperature and refluxed for 2 h then cooling to room temperature. IONPs were obtained with different morphology by controlling the storage duration of iron oleate, the amount of sodium oleate, 1-octadecene and heating process. For more synthesis details see our previously published paper.37 The products were separated by centrifugation with the addition of isopropanol, washed with ethanol for two times, and then re-dispersed in hexane for storage. For cell culture, 50 mg of as-prepared nanoparticles and 100 mg of sodium citrate were dissolved in a mixture containing 4 mL of hexane, 4 mL of water, and 6 mL of acetone. Then the solution was stirred at 70 °C for ligand exchange process. IONPs were collected by centrifugation and washed with acetone before finally dispersed in water.

Mice. Nlrp3-/-, ASC-/- and Caspase1-/- mice in C57BL/6 background were kindly provided by Professor Zhou Rongbin (University of Science and Technology of China, Hefei, China). Protocols for animal experimentation were reviewed and approved by the Ethics Committee of University of Science and Technology of China.

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Cell culture. The culture of BMDM cells and human THP-1 cells were described in previously published paper.38-39

Inflammasome activation. BMDM cells were primed with LPS (500 ng/ml) in opti-MEM for 3 hr, followed by treatment with different nanomaterials for 6 hr. Collection of culture supernatant for ELISA while immunoblotting was performed for cell lysates.

Cytotoxicity assay. Determination of cell viability by CCK-8 assay. Cells were seeded into plates, followed by treatment with different IONPs. Then cells were treated with CCK-8 (1:10) for 2-4 hours. The optical density (O.D.) at 450 nm wavelength was determined.

ELISA assay. Culture supernatants from treated cells were collected after centrifugation and then assayed for TNF-α and IL-1β according to manufacturer’s instructions.

ROS

detection.

Measurement

of

intracellular

ROS

production

by

2',7'-dichlorofluorescein diacetate (DCFH-DA). After treatment with different IONPs, cells were incubated for 30 min with 10 μM DCFH-DA in DMEM without antibody and FBS. Cells after being washed with PBS were visualized by fluorescence microscope.

Lysosome Damage Assessment. After treatment with different IONPs, cells were incubated for 20-30 min with Lysotracker Red or Lysosensor Green in DMEM 7



without antibody and FBS. Cells after being washed with PBS were visualized by fluorescence microscope. The mean fluorescence intensity was determined by flow cytometer (BD Bioscience), and the data were analyzed by FlowJo software.

Intracellular Fe measurements. Determination of intracellular Fe concentration through inductively coupled plasma mass spectrometry (ICP-MS) with a PerkinElmer Optima 2000DV spectrometer. Cells treated with various IONPs were washed with sterile PBS to remove the excess particles and were then digested by boiling in ultrapure HNO3 for 1 hour before sending off for ICP analysis.

Visualization of caspase 1 activation. Caspase 1 activation was visualized by FAM-FLICA Caspase-1 Assays kit. Cells were seeded into plates, followed by treatment with different shape of IONPs. The supernatants were removed and used PBS for wash cell 3 times then stained with FAM-YVAD-FMK (green), Propidium iodide (red) and Hoechst (blue). After washing off the excess dyes with sterile PBS, cells were visualized under fluorescent microscope.

Immunoblotting. Culture supernantants and cell lysate were separated by electrophoresis on SDS-PAGE and transferred to NC membranes. Blocking with 5% bovine serum albumin for 1 hr, the membranes were sequentially incubated with primary antibody and secondary antibody. Use HRP Substrate to detect the protein bands. The images of the protein bands were analyzed by ImageJ software.

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Potassium efflux assay. After the culture media were thoroughly removed, cells were collected and then digested by boiling in ultrapure HNO3 for 1 hour, followed by K+ measurements using ICP-MS.

Statistical analysis. All values were expressed as the Mean ± SEM, and analyzed by two-tailed Student's t-tests. *P

Impact of Morphology on Iron Oxide Nanoparticles-Induced

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