Distribution of SiO2 nanoparticles in 3D liver microtissues

Introduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation

Ein Zelllinien-basiertes Co-Kulturmodell der entzündeten Darmmukosa und dessen Anwendung für Sicherheits- und Wirksamkeitsstudien von Nanopartikeln

In vitro cell culture models are an important alternative for animal testing, as they are less ethically questionable and avoid the problem of non-representative results due to species differences. A previously established 3D co-culture model of the inflamed intestinal mucosa consisting of human epithelial and primary immune cells was further developed by replacing primary cells by two human monocytic cell lines in order to have a stable and more reproducible system, which shows a well preserved ultrastructure and good barrier properties. With this model the safety of different nanoparticles (NPs) was investigated and it was shown that Ag NPs show a high cytotoxicity. Co-culture models incubated with Au NPs showed an inflammatory response although no toxic effects were measurable. Furthermore, differences between inflamed and non-inflamed co-cultures and Caco 2 monocultures were shown with the result that Caco 2 cells are more sensitive to toxic Ag NPs than the co-culture models. For the treatment of inflammatory bowel disease (IBD) nano- and microparticulate drug delivery systems (DDS) were developed, containing anti-inflammatory compounds budesonide and cyclosporine A. The anti-inflammatory effect of the formulations on the inflamed co-culture was shown by TEER and IL-8 measurement. In summary, the further developed co-culture model is a useful tool for safety testing of nanomaterials and can be used to test anti-inflammatory effects of DDS for the treatment of IBD.In vitro Modelle sind eine wichtige Alternative zu Tierversuchen, da sie ethisch weniger fragwürdig sind und nicht-repräsentative Ergebnisse aufgrund von Spezies-Unterschieden vermeiden. Ein 3D Co-Kultur Modell der entzündeten Darmmukosa, bestehend aus menschlichen Epithel- und primären Immunzellen wurde entworfen, welches in der vorliegenden Arbeit weiterentwickelt wurde, indem die Primärzellen durch Zelllinien ersetzt wurden, um ein reproduzierbareres Modell zu erhalten. Mit diesem Modell wurde die Sicherheit verschiedener Nanomaterialen untersucht und es wurde gezeigt, dass Ag NP zytotoxisch wirken. Zell-Modelle, inkubiert mit Au NP, zeigten eine entzündliche Antwort, obwohl keine Toxizität gemessen werden konnte. Zusätzlich wurde gezeigt, dass Caco-2 Monokulturen empfindlicher gegenüber toxischen Ag NP sind als die Co-Kultur Modelle. Für die Behandlung entzündlicher Darmerkrankungen wurden nano- und mikropartikuläre Arzneistoffträgersysteme entwickelt, die die anti-entzündlichen Verbindungen Budesonide und Ciclosporin A enthalten. Der anti-entzündliche Effekt dieser Partikel wurde im entzündeten Co-Kultur Modell des Darms durch TEER und Zytokin Messungen gezeigt. Dies zeigt: Das weiterentwickelte Co-Kultur Modell ist ein nützliches Werkzeug für Sicherheitstests von Nanomaterialien. Darüber hinaus kann es genutzt werden, um anti-entzündliche Effekte von Arzneistoffträgersystemen für die Behandlung entzündlicher Darmerkrankungen zu testen

Transfection System of Amino-Functionalized Calcium Phosphate Nanoparticles: In Vitro Efficacy, Biodegradability, and Immunogenicity Study

Many methods have been developed in order to use calcium phosphate (CaP) for delivering nucleotides into living cells. Surface functionalization of CaP nanoparticles (CaP NPs) with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane was shown recently to achieve dispersed NPs with a positive surface charge, capable of transfection (<i>Chem. Mater.</i> <b>2013</b>, <i>25</i> (18), 3667). In this study, different crystal structures of amino-modified CaP NPs (brushite and hydroxyapatite) were investigated for their interaction in cell culture systems in more detail. Qualitative (confocal laser scanning microscopy) and quantitative (flow cytometry) transfection experiments with two cell lines showed the higher transfection efficacy of brushite versus hydroxyapatite. The transfection also revealed a cell type dependency. HEK293 cells were easier to transfect compared to A549 cells. This result was supported by the cytotoxicity results. A549 cells showed a higher degree of tolerance toward the CaP NPs. Further, the impact of the surface modification on the interaction with macrophages and complement as two important components of the innate immune system were considered. The amine surface functionalization had an effect of decreasing the release of proinflammatory cytokines. The complement interaction investigated by a C3a complement activation assay did show no significant differences between CaP NPs without or with amine modification and overall weak interaction. Finally, the degradation of CaP NPs in biological media was studied with respect to the two crystal structures and at acidic and neutral pH. Both amino-modified CaP NPs disintegrate within days at neutral pH, with a notable faster disintegration of brushite NPs at acidic pH. In summary, the fair transfection capability of this amino functionalized CaP NPs together with the excellent biocompatibility, biodegradability, and low immunogenicity make them interesting candidates for further evaluation

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials.

Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO2, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO2 or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation

Silica-coated Au@ZnO Janus particles and their stability in epithelial cells

Multicomponent particles have emerged in recent years as new compartmentalized colloids with two sides of different chemistry or polarity that have opened up a wide field of unique applications in medicine, biochemistry, optics, physics and chemistry. A drawback of particles containing a ZnO hemisphere is their low stability in biological environment due to the amphoteric properties of Zn2 . Therefore we have synthesized monodisperse Au@ZnO Janus particles by seed mediated nucleation and growth whose ZnO domain was coated selectively with a thin SiO2 layer as a protection from the surrounding environment that imparts stability in aqueous media while the Au domain remained untouched. The thickness of the SiO2 layer could be precisely controlled. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g. antibodies, proteins) in a single step for converting the photoluminescent and photocatalytic active Janus nanoparticles into multifunctional efficient vehicles for cell targeting. The SiO2-coated functionalized nanoparticles were stable in buffer solutions and other aqueous systems. Biocompatibility and potential biomedical applications of the Au@ZnO@SiO2 Janus particles were assayed by a cell viability analysis by co-incubating the Au@ZnO@SiO2 Janus particles with epithelia cells and compared to those of uncoated ZnO. E