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Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

ABSTRACT: Modulating biomolecular networks in cells with peptides and proteins has become a promising therapeutic strategy and effective biological tools. A simple and effective reagent that can bring functional proteins into cells can increase efficacy and allow more investigations. Here we show that the relatively non-toxic and non-immunogenic oxidized carbon black particles (OCBs) prepared from commercially available carbon black can deliver a 300?kDa protein directly into cells, without an involvement of a cellular endocytosis. Experiments with cell-sized liposomes indicate that OCBs directly interact with phospholipids and induce membrane leakages. Delivery of human monoclonal antibodies (HuMAbs, 150?kDa) with specific affinity towards dengue viruses (DENV) into DENV-infected Vero cells by OCBs results in HuMAbs distribution all over cells' interior and effective viral neutralization. An ability of OCBs to deliver big functional/therapeutic proteins into cells should open doors for more protein drug investigations and new levels of antibody therapies and biological studies.

SUBMITTER: Amornwachirabodee K

PROVIDER: S-EPMC5802750 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

Amornwachirabodee Kittima K Tantimekin Nattapol N Pan-In Porntip P Palaga Tanapat T Pienpinijtham Prompong P Pipattanaboon Chonlatip C Sukmanee Thanyada T Ritprajak Patcharee P Charoenpat Promchat P Pitaksajjakul Pannamthip P Ramasoota Pongrama P Wanichwecharungruang Supason S

Scientific reports 20180206 1

Modulating biomolecular networks in cells with peptides and proteins has become a promising therapeutic strategy and effective biological tools. A simple and effective reagent that can bring functional proteins into cells can increase efficacy and allow more investigations. Here we show that the relatively non-toxic and non-immunogenic oxidized carbon black particles (OCBs) prepared from commercially available carbon black can deliver a 300 kDa protein directly into cells, without an involvement ...[more]

PMID: 29410523

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Project description:Rationale: The adverse health effects of nano-particulate pollutants have attracted much attention in recent years. Carbon nanomaterials are recognized as risk factors for prolonged inflammatory responses and diffuse alveolar injury. Previous research indicated a central role of alveolar macrophages in the pathogenesis of particle-related lung disease, but the underlying mechanism remains largely unknown. Methods: C57BL/6 mice were intratracheally instilled with carbon black nanoparticles (CBNPs). Cell necrosis and the infiltrated neutrophils in the lungs were detected by flow cytometry. Release of mitochondria was observed with Mito Tracker and mitochondrial DNA (mtDNA) was quantified by qPCR via Taqman probes. TLR9-p38 MAPK signaling pathway was detected by Western blotting. The production of lipid chemoattractant leukotriene B4 (LTB4) in the supernatant and bronchoalveolar lavage fluid (BALF) was quantitated using an enzyme immunoassay (EIA). Results: In the present study, we found that a single instillation of CBNPs induced neutrophil influx in C57BL/6 mice as early as 4 h post-exposure following the rapid appearance of cell damage indicators in BALF at 30 min. Macrophages exposed to CBNPs showed necrotic features and were characterized by lysosome rupture, cathepsin B release, reactive oxygen species generation, and reduced intracellular ATP level. Necrosis was partly inhibited by a specific lysosomal cathepsin B inhibitor CA074 Me. Further analyses suggested that the resulting leakage of mtDNA from the necrotic cells activated neutrophils and triggered severe inflammation in vivo. Pulmonary neutrophilic inflammation induced by mtDNA was reduced in TLR9-/- mice. Additionally, mtDNA induced LTB4 production from macrophages, which may contribute to neutrophil recruitment. Conclusion: We demonstrated here that CBNPs induce acute cell necrosis through lysosomal rupture and that mtDNA released from necrotic cells functions as a key event mediating pulmonary neutrophilic inflammation. This study described a novel aspect of the pathogenesis of particle-induced inflammatory response and provided a possible therapeutic target for the regulation of inflammation.

Project description:Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 μg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage.

Dataset Information

Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

Publications

Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

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