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Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches

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A special issue of Nanomaterials (ISSN 2079-4991).

Deadline for manuscript submissions: closed (31 October 2017) | Viewed by 69382

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Dr. Martin Wiemann

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IBE R and D gGmbH, Institute for Lung Health, Mendelstrasse 11, D-48149 Münster, Germany
Interests: effects of (nano)particle and fibers on the lung; general particle toxicity on human and animal tissue; in vitro studies on cells to predict particle effects; macrophage biology

Special Issue Information

Dear Colleagues,

Within the last decade, thousands of scientific publications have investigated the biologic activity of nanoparticles using mammalian cell models and rodents for risk estimation. However, studies in which in vitro and in vivo experiments make use of essentially the same nanoparticles have been undertaken far less frequently, although a maximal degree of similarity between in vitro and in vivo approaches appears mandatory for a sound validation of in vitro results.

In this sense, the in vivo findings based on biokinetic behaviour of nanoparticles and on toxicological results should set out the frame for the design of predictive cell tests: Starting with the selection of most appropriate and representative test cells, meaningful cellular doses of nanoparticles appear most important, along with a fine-tuned experimental setting meant to unravel and understand the sophisticated response of cells upon nanoparticle treatment.

This Special Issue of Nanomaterials is therefore devoted to original contributions in which in vitro work is reasoned and accompanied by in vivo studies with the aim to maximize the degree of similarity of both approaches. Well-motivated studies aiming at the risk estimation for animals and humans are welcome and acceptance will not depend on the degree of success, as long as contributions are in accord with 3R principles.

Prof. Dr. Martin Wiemann
Guest Editor

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Keywords

Nanotoxicology
in vivo–in vitro comparison
Inhalation
intratracheal instillation
3R principle
validation study

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15 pages, 4423 KiB

Open AccessFeature PaperArticle

Detection of ZrO₂ Nanoparticles in Lung Tissue Sections by Time-of-Flight Secondary Ion Mass Spectrometry and Ion Beam Microscopy

by Lothar Veith, Julia Böttner, Antje Vennemann, Daniel Breitenstein, Carsten Engelhard, Jan Meijer, Irina Estrela-Lopis, Martin Wiemann and Birgit Hagenhoff

Nanomaterials 2018, 8(1), 44; https://doi.org/10.3390/nano8010044 - 15 Jan 2018

Cited by 14 | Viewed by 6387

Abstract

The increasing use of nanoparticles (NP) in commercial products requires elaborated techniques to detect NP in the tissue of exposed organisms. However, due to the low amount of material, the detection and exact localization of NP within tissue sections is demanding. In this respect, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Ion Beam Microscopy (IBM) are promising techniques, because they both offer sub-micron lateral resolutions along with high sensitivities. Here, we compare the performance of the non-material-consumptive IBM and material-consumptive ToF-SIMS for the detection of ZrO₂ NP (primary size 9–10 nm) in rat lung tissue. Unfixed or methanol-fixed air-dried cryo-sections were subjected to IBM using proton beam scanning or to three-dimensional ToF-SIMS (3D ToF-SIMS) using either oxygen or argon gas cluster ion beams for complete sample sputtering. Some sample sites were analyzed first by IBM and subsequently by 3D ToF-SIMS, to compare results from exactly the same site. Both techniques revealed that ZrO₂ NP particles occurred mostly agglomerated in phagocytic cells with only small quantities being associated to the lung epithelium, with Zr, S, and P colocalized within the same biological structures. However, while IBM provided quantitative information on element distribution, 3D ToF-SIMS delivered a higher lateral resolution and a lower limit of detection under these conditions. We, therefore, conclude that 3D ToF-SIMS, although not yet a quantitative technique, is a highly valuable tool for the detection of NP in biological tissue. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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11960 KiB

Open AccessFeature PaperArticle

Silver Nanoparticles in the Lung: Toxic Effects and Focal Accumulation of Silver in Remote Organs

by Martin Wiemann, Antje Vennemann, Franziska Blaske, Michael Sperling and Uwe Karst

Nanomaterials 2017, 7(12), 441; https://doi.org/10.3390/nano7120441 - 12 Dec 2017

Cited by 69 | Viewed by 8072

Abstract

The distribution of silver (Ag) into remote organs secondary to the application of Ag nanoparticles (Ag-NP) to the lung is still incompletely understood and was investigated in the rat with imaging methods. Dose-finding experiments were carried out with 50 nm- or 200 nm-sized polyvinyl pyrrolidine (PVP)-coated Ag-NP using alveolar macrophages in vitro and female rats, which received Ag-NP via intratracheal instillation. In the main study, we administered 37.5–300 µg per rat lung of the more toxic Ag50-PVP and assessed the broncho-alveolar lavage fluid (BALF) for inflammatory cells, total protein and fibronectin after three and 21 days. In parallel, lung tissue was analysed for DNA double-strand breaks and altered cell proliferation. While 75–150 µg Ag50-PVP per rat lung caused a reversible inflammation, 300 µg led to DNA damage, accelerated cell proliferation and progressively increasing numbers of neutrophilic granulocytes. Ag accumulation was significant in homogenates of liver and other peripheral organs upon lung dose of ≥75 µg. Quantitative laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) combined with enhanced dark field microscopy and autometallography revealed focal accumulations of Ag and/or Ag-NP in sections of peripheral organs: mediastinal lymph nodes contained Ag-NP especially in peripheral macrophages and Ag in argyrophilic fibres. In the kidney, Ag had accumulated within proximal tubuli, while renal filter structures contained no Ag. Discrete localizations were also observed in immune cells of liver and spleen. Overall, the study shows that concentrations of Ag-NP, which elicit a transient inflammation in the rat lung, lead to focal accumulations of Ag in peripheral organs, and this might pose a risk to particular cell populations in remote sites. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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1864 KiB

Open AccessArticle

Improved In Vivo Efficacy of Anti-Hypertensive Biopeptides Encapsulated in Chitosan Nanoparticles Fabricated by Ionotropic Gelation on Spontaneously Hypertensive Rats

by Shehu Muhammad Auwal, Mohammad Zarei, Chin Ping Tan, Mahiran Basri and Nazamid Saari

Nanomaterials 2017, 7(12), 421; https://doi.org/10.3390/nano7120421 - 2 Dec 2017

Cited by 35 | Viewed by 5323

Abstract

Recent biotechnological advances in the food industry have led to the enzymatic production of angiotensin I-converting enzyme (ACE)-inhibitory biopeptides with a strong blood pressure lowering effect from different food proteins. However, the safe oral administration of biopeptides is impeded by their enzymatic degradation due to gastrointestinal digestion. Consequently, nanoparticle (NP)-based delivery systems are used to overcome these gastrointestinal barriers to maintain the improved bioavailability and efficacy of the encapsulated biopeptides. In the present study, the ACE-inhibitory biopeptides were generated from stone fish (Actinopyga lecanora) protein using bromelain and stabilized by their encapsulation in chitosan (chit) nanoparticles (NPs). The nanoparticles were characterized for in vitro physicochemical properties and their antihypertensive effect was then evaluated on spontaneously hypertensive rats (SHRs). The results of a physicochemical characterization showed a small particle size of 162.70 nm, a polydispersity index (pdi) value of 0.28, a zeta potential of 48.78 mV, a high encapsulation efficiency of 75.36%, a high melting temperature of 146.78 °C and an in vitro sustained release of the biopeptides. The results of the in vivo efficacy indicated a dose-dependent blood pressure lowering effect of the biopeptide-loaded nanoparticles that was significantly higher (p < 0.05) compared with the un-encapsulated biopeptides. Moreover, the results of a morphological examination using transmission electron microscopy (TEM) demonstrated the nanoparticles as homogenous and spherical. Thus, the ACE-inhibitory biopeptides stabilized by chitosan nanoparticles can effectively reduce blood pressure for an extended period of time in hypertensive individuals. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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21602 KiB

Open AccessFeature PaperArticle

Specific Surface Modifications of Silica Nanoparticles Diminish Inflammasome Activation and In Vivo Expression of Selected Inflammatory Genes

by Viviana Marzaioli, Christina J. Groß, Ingrid Weichenmeier, Carsten B. Schmidt-Weber, Jan Gutermuth, Olaf Groß and Francesca Alessandrini

Nanomaterials 2017, 7(11), 355; https://doi.org/10.3390/nano7110355 - 30 Oct 2017

Cited by 16 | Viewed by 5108

Abstract

Silica (SiO₂) nanoparticles (NPs) usage includes, but is not limited to, industrial and biomedical applications. Toxic effects of SiO₂ NPs have been explored either in vitro or in vivo, assessing different surface modifications to reduce their harmful effects. Here, murine bone marrow-derived dendritic (BMDC) and a mouse model of mild allergic inflammation were used to study inflammasome activation and lung inflammation. Our results showed that SiO₂ plain NPs induced NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation, increasing interleukin (IL)-1β release in vitro, and, to a lesser extent, in vivo. In addition, SiO₂ plain NPs triggered a pulmonary inflammatory milieu in both non-sensitized (NS) and sensitized (S) mice, by inducing the expression of key inflammatory cytokines and chemokines. Electron microscopy showed that SiO₂ NPs were mostly localized in alveolar macrophages, within vesicles and/or in phagolysosomes. Both the in vitro and the in vivo effects of SiO NPs were attenuated by coating NPs with phosphonate or amino groups, whereas PEGylation, although it mitigated inflammasome activation in vitro, was not a successful coating strategy in vivo. These findings highlight that multiple assays are required to determine the effect of surface modifications in limiting NPs inflammatory potential. Taken together, these data are obtained by comparing in vitro and in vivo effects of SiO₂ NPs suggest the use of amino and phosphonate coating of silica NPs for commercial purposes and targeted applications, as they significantly reduce their proinflammatory potential. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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3486 KiB

Open AccessArticle

Endocytosis of Corn Oil-Caseinate Emulsions In Vitro: Impacts of Droplet Sizes

by Yuting Fan, Yuzhu Zhang, Wally Yokoyama and Jiang Yi

Nanomaterials 2017, 7(11), 349; https://doi.org/10.3390/nano7110349 - 26 Oct 2017

Cited by 34 | Viewed by 5549

Abstract

The relative uptake and mechanisms of lipid-based emulsions of three different particle diameters by Caco-2 cells were studied. The corn oil-sodium caseinate emulsions showed little or no cytotoxicity even at 2 mg/mL protein concentration for any of the three droplet size emulsions. Confocal laser scanning microscopy (CLSM) of Nile red containing emulsions showed that the lipid-based emulsions were absorbed by Caco-2 cells. A negative correlation between the mean droplet size and cellular uptake was observed. There was a time-dependent and energy-dependent uptake as shown by incubation at different times and treatment with sodium azide a general inhibitor of active transport. The endocytosis of lipid-based emulsions was size-dependent. The internalization of nanoemulsion droplets into Caco-2 cells mainly occurred through clathrin- and caveolae/lipid raft-related pathways, while macropinocytosis route played the most important role for 556 nm emulsion endocytosis as shown by the use of specific pathway inhibitors. Permeability of the emulsion through the apical or basal routes also suggested that active transport may be the main route for lipid-based nanoemulsions. The results may assist in the design and application of lipid-based nanoemulsions in nutraceuticals and pharmaceuticals delivery. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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3550 KiB

Open AccessArticle

Pro-Inflammatory versus Immunomodulatory Effects of Silver Nanoparticles in the Lung: The Critical Role of Dose, Size and Surface Modification

by Francesca Alessandrini, Antje Vennemann, Silvia Gschwendtner, Avidan U. Neumann, Michael Rothballer, Tanja Seher, Maria Wimmer, Susanne Kublik, Claudia Traidl-Hoffmann, Michael Schloter, Martin Wiemann and Carsten B. Schmidt-Weber

Nanomaterials 2017, 7(10), 300; https://doi.org/10.3390/nano7100300 - 29 Sep 2017

Cited by 48 | Viewed by 6502

Abstract

The growing use of silver nanoparticles (Ag-NPs) in consumer products raises concerns about their toxicological potential. The purpose of the study was to investigate the size- and coating-dependent pulmonary toxicity of Ag-NPs in vitro and in vivo, using an ovalbumin (OVA)-mouse allergy model. Supernatants from (5.6–45 µg/mL) Ag50-PVP, Ag200-PVP or Ag50-citrate-treated NR8383 alveolar macrophages were tested for lactate dehydrogenase and glucuronidase activity, tumor necrosis factor (TNF)-α release and reactive oxygen species (ROS) production. For the in vivo study, NPs were intratracheally instilled in non-sensitized (NS) and OVA-sensitized (S) mice (1–50 µg/mouse) prior to OVA-challenge and bronchoalveolar lavage fluid (BALF) inflammatory infiltrate was evaluated five days after challenge. In vitro results showed a dose-dependent cytotoxicity of Ag-NPs, which was highest for Ag50-polyvinilpyrrolidone (PVP), followed by Ag50-citrate, and lowest for Ag200-PVP. In vivo 10–50 µg Ag50-PVP triggered a dose-dependent pulmonary inflammatory milieu in NS and S mice, which was significantly higher in S mice and was dampened upon instillation of Ag200-PVP. Surprisingly, instillation of 1 µg Ag50-PVP significantly reduced OVA-induced inflammatory infiltrate in S mice and had no adverse effect in NS mice. Ag50-citrate showed similar beneficial effects at low concentrations and attenuated pro-inflammatory effects at high concentrations. The lung microbiome was altered by NPs instillation dependent on coating and/or mouse batch, showing the most pronounced effects upon instillation of 50 µg Ag50-citrate, which caused an increased abundance of operational taxonomic units assigned to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, no correlation with the biphasic effect of low and high Ag-NPs dose was found. Altogether, both in vitro and in vivo data on the pulmonary effects of Ag-NPs suggest the critical role of the size, dose and surface functionalization of Ag-NPs, especially in susceptible allergic individuals. From the perspective of occupational health, care should be taken by the production of Ag-NPs-containing consumer products. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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14402 KiB

Open AccessArticle

Differential Effects of Surface-Functionalized Zirconium Oxide Nanoparticles on Alveolar Macrophages, Rat Lung, and a Mouse Allergy Model

by Antje Vennemann, Francesca Alessandrini and Martin Wiemann

Nanomaterials 2017, 7(9), 280; https://doi.org/10.3390/nano7090280 - 19 Sep 2017

Cited by 23 | Viewed by 6112

Abstract

Nanoparticles (NPs) may affect the lung via their chemical composition on the surface. Here, we compared the bioactivity of zirconium oxide (ZrO₂) NPs coated with either aminopropilsilane (APTS), tetraoxidecanoic acid (TODS), polyethyleneglycol (PGA), or acrylic acid (Acryl). Supernatants from NPs-treated cultured alveolar macrophages (NR8383) tested for lactate dehydrogenase, glucuronidase, tumor necrosis factor α, and H₂O₂ formation revealed dose-dependent effects, with only gradual differences among particles whose gravitational settling and cellular uptake were similar. We selected TODS- and Acryl-coated NPs for intratracheal administration into the rat lung. Darkfield and hyperspectral microscopy combined with immunocytochemistry showed that both NPs qualities accumulate mainly within the alveolar macrophage compartment, although minute amounts also occurred in neutrophilic granulocytes. Dose-dependent signs of inflammation were found in the broncho-alveolar lavage fluid on day 3 but no longer on day 21 post-application of ≥1.2 mg per lung; again only minor differences occurred between TODS- and Acryl-coated NPs. In contrast, the response of allergic mice was overall higher compared to control mice and dependent on the surface modification. Increases in eosinophils, lymphocytes and macrophages were highest following ZrO₂-PGA administration, followed by ZrO₂-Acryl, ZrO₂-TODS, and ZrO₂-APTS. We conclude that surface functionalization of ZrO₂ NPs has minor effects on the inflammatory lung response of rats and mice, but is most relevant for an allergic mouse model. Allergic individuals may therefore be more susceptible to exposure to NPs with specific surface modifications. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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6096 KiB

Open AccessArticle

Near-Infrared-Triggered Photodynamic Therapy toward Breast Cancer Cells Using Dendrimer-Functionalized Upconversion Nanoparticles

by Bing-Yen Wang, Ming-Liang Liao, Guan-Ci Hong, Wen-Wei Chang and Chih-Chien Chu

Nanomaterials 2017, 7(9), 269; https://doi.org/10.3390/nano7090269 - 11 Sep 2017

Cited by 47 | Viewed by 7846

Abstract

Water-soluble upconversion nanoparticles (UCNPs) that exhibit significant ultraviolet, blue, and red emissions under 980-nm laser excitation were successfully synthesized for performing near infrared (NIR)-triggered photodynamic therapy (PDT). The lanthanide-doped UCNPs bearing oleate ligands were first exchanged by citrates to generate polyanionic surfaces and then sequentially encapsulated with NH₂-terminated poly(amido amine) (PAMAM) dendrimers (G4) and chlorine6 (Ce6) using a layer-by-layer (LBL) absorption strategy. Transmission electron microscopy and X-ray diffraction analysis confirm that the hybrid UCNPs possess a polygonal morphology with an average dimension of 16.0 ± 2.1 nm and α-phase crystallinity. A simple calculation derived through thermogravimetric analysis revealed that one polycationic G4 dendrimer could be firmly accommodated by approximately 150 polyanionic citrates through multivalent interactions. Moreover, zeta potential measurements indicated that the LBL fabrication results in the hybrid nanoparticles with positively charged surfaces originated from these dendrimers, which assist the cellular uptake in biological specimens. The cytotoxic singlet oxygen based on the photosensitization of the adsorbed Ce6 through the upconversion emissions can be readily accumulated by increasing the irradiation time of the incident lasers. Compared with that of 660-nm lasers, NIR-laser excitation exhibits optimized in vitro PDT effects toward human breast cancer MCF-7 cells cultured in the tumorspheres, and less than 40% of cells survived under a low Ce6 dosage of 2.5 × 10⁻⁷ M. Fluorescence microscopy analysis indicated that the NIR-driven PDT causes more effective destruction of the cells located inside spheres that exhibit significant cancer stem cell or progenitor cell properties. Moreover, an in vivo assessment based on immunohistochemical analysis for a 4T1 tumor-bearing mouse model confirmed the effective inhibition of cancer cell proliferation through cellular DNA damage by the expression of Ki67 and γH2AX^ser139 protein markers. Thus, the hybrid UCNPs are a promising NIR-triggered PDT module for cancer treatment. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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2950 KiB

Open AccessArticle

Encapsulation of 16-Hydroxycleroda-3,13-Dine-16,15-Olide in Mesoporous Silica Nanoparticles as a Natural Dipeptidyl Peptidase-4 Inhibitor Potentiated Hypoglycemia in Diabetic Mice

by Po-Kai Huang, Shi-Xiang Lin, May-Jywan Tsai, Max K. Leong, Shian-Ren Lin, Ranjith Kumar Kankala, Chia-Hung Lee and Ching-Feng Weng

Nanomaterials 2017, 7(5), 112; https://doi.org/10.3390/nano7050112 - 12 May 2017

Cited by 28 | Viewed by 6233

Abstract

Natural supplements comprise good efficacy with less adverse effects as against diabetic therapy, but their advancement as anti-diabetic agents is unsatisfactory with regard to the delivery system. Dipeptidyl peptidase-4 (DPP4)/CD26) can degrade glucagon-like pepetide-1 (GLP-1) which renders a decrease of blood glucose levels. 16-hydroxycleroda-3,13-dine-16,15-olide (HCD) extracted from Polyalthia longifolia, exhibits numerous medicinal potentials including hypoglycemic potential. On consideration of HCD application, the bioavailability is affected by low solubility. Extended experiments of anti-diabetic efficacy confirmed HCD biocompatible with mesoporous silica nanoparticles (MSNs) encapsulation resulted in a sustained release property in delivering HCD for the inhibition of DPP4 via the activity and protein levels of DPP4 analysis. In the enzymatic activity assay, MSN-HCD directly changed DPP4 activity. Moreover, MSN-HCD nanoparticles were treated with Caco-2 cells and the protein levels of DPP4 determined within the cells. The results revealed that MSN-HCD caused reduction of DPP4 activity in a time- and dose-dependent fashion. Orally administered MSN-HCD in diet-induced diabetic mice alleviated blood glucose via an oral glucose tolerance test. In addition, administration of MSN-HCD for five weeks revealed that the biochemical cues such as pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), triglycerides (TG), cholesterol (CHO), and glycated hemoglobin (HbA1c) in mice were commendable as further confirmation of MSN-HCD efficacy and less adverse effects in down-regulation of hyperglycemia. Furthermore, this formulation effectively controlled blood glucose and significantly decreased the body weight of mice, suggesting that MSN-HCD exerts natural DPP4 inhibitor as a potential clinical drug for the treatment of diabetes. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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Review

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785 KiB

Open AccessReview

Potential Toxicity and Underlying Mechanisms Associated with Pulmonary Exposure to Iron Oxide Nanoparticles: Conflicting Literature and Unclear Risk

by Tiffany G. Kornberg, Todd A. Stueckle, James A. Antonini, Yon Rojanasakul, Vincent Castranova, Yong Yang and Liying Wang

Nanomaterials 2017, 7(10), 307; https://doi.org/10.3390/nano7100307 - 6 Oct 2017

Cited by 63 | Viewed by 9354

Abstract

Abstract: Fine/micron-sized iron oxide particulates are incidentally released from a number of industrial processes, including iron ore mining, steel processing, welding, and pyrite production. Some research suggests that occupational exposure to these particulates is linked to an increased risk of adverse respiratory outcomes, whereas other studies suggest that iron oxide is biologically benign. Iron oxide nanoparticles (IONPs), which are less than 100 nm in diameter, have recently surged in use as components of novel drug delivery systems, unique imaging protocols, as environmental catalysts, and for incorporation into thermoplastics. However, the adverse outcomes associated with occupational exposure to IONPs remain relatively unknown. Relevant in vivo studies suggest that pulmonary exposure to IONPs may induce inflammation, pulmonary fibrosis, genotoxicity, and extra-pulmonary effects. This correlates well with in vitro studies that utilize relevant dose, cell type(s), and meaningful end points. A majority of these adverse outcomes are attributed to increased oxidative stress, most likely caused by particle internalization, dissolution, release of free iron ions, and disruption of iron homeostasis. However, because the overall toxicity profile of IONPs is not well understood, it is difficult to set safe exposure limit recommendations that would be adequate for the protection of at-risk workers. This review article will focus on known risks following IONPs exposure supported by human, animal, and cell culture-based studies, the potential challenges intrinsic to IONPs toxicity assessment, and how these may contribute to the poorly characterized IONPs toxicity profile. Full article

(This article belongs to the Special Issue Nanoparticles in Vivo and in Vitro Studies: A Collection of Parallel Approaches)

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