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Biocompatibility and biodistribution of functionalized carbon nano-onions (f-CNOs) in a vertebrate model

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ABSTRACT

Functionalized carbon nano-onions (f-CNOs) are of great interest as platforms for imaging, diagnostic and therapeutic applications due to their high cellular uptake and low cytotoxicity. To date, the toxicological effects of f-CNOs on vertebrates have not been reported. In this study, the possible biological impact of f-CNOs on zebrafish during development is investigated, evaluating different toxicity end-points such as the survival rate, hatching rate, and heart beat rate. Furthermore, a bio-distribution study of boron dipyrromethene (BODIPY) functionalized CNOs in zebrafish larvae is performed by utilizing inverted selective plane illumination microscopy (iSPIM), due to its intrinsic capability of allowing for fast 3D imaging. Our in vivo findings indicate that f-CNOs exhibit no toxicity, good biocompatibility (in the concentration range of 5–100 μg mL−1) and a homogenous biodistribution in zebrafish larvae.

No MeSH data available.


(a) Schematic representation of benz-CNOs (left) and BODIPY-CNOs (right); Blue, nitrogen atom (N); pink, boron atom (B); green, fluorine atom (F). (b) HR-TEM image of BODIPY-CNOs.
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f1: (a) Schematic representation of benz-CNOs (left) and BODIPY-CNOs (right); Blue, nitrogen atom (N); pink, boron atom (B); green, fluorine atom (F). (b) HR-TEM image of BODIPY-CNOs.

Mentions: For any nanomaterial utilized in an application that might lead to a release to the environment or to the exposure of living beings, in particular humans, an accurate risk assessment as well as a toxicological screening is highly needed2728. The aquatic environment is of high importance since any contamination of water might lead to a wide distribution of the contaminant and thus to major pollution. All the biological in vitro studies investigating the effects of small CNOs (diameter of approx. 5 nm) with different surface functionalization on a variety of different cell cultures describe CNOs as a highly biocompatible nanomaterial. The effect of large CNOs (diameter of approx. 30 nm) on the immune system indicates that the cell response is highly dependent on the structure29. The inflammatory potential of small f-CNOs on immortalized bone-marrow-derived mouse macrophages and mouse bone-marrow-derived dendritic cells is found to be negligible and significantly lower than the effects of similarly functionalized single wall carbon nanotubes12. A recent report comparing large CNOs (diameter of 50–100 nm) with multiwall carbon nanotubes corroborates these findings30. We recently showed that small functionalized CNOs have no significant adverse effects on three weeks old freshwater polyp Hydra vulgaris31. The results reported on this very simple and basal animal, in addition to in vitro studies, suggest that CNO is a biocompatible and safe nanomaterial, but are not sufficient to rule out possible risks of CNOs exposure and release. Additional long-term toxicity studies of CNOs on complex organisms, as well as the fate of CNOs in biological systems, are strongly needed. Zebrafish represents an emerging and excellent model organism, mainly due to the fact that it has a remarkable similarity in the molecular signalling processes, cellular structure, anatomy and physiology to other higher order vertebrates323334353637. Moreover, zebrafish embryos are ideal for high-throughput screening due to their external development, optical transparency, and short breeding cycle3839. Zebrafish development represents a valuable tool to assess the in vivo toxicity and biocompatibility of drugs, chemicals and nanomaterials, with a focus on the developmental effects, and to obtain toxicity information at the whole animal level404142434445. Several approaches using zebrafish embryos or larvae as an animal model have been developed in recent years to assess embryonic effects of chemicals, drugs and nanomaterials464748, in order to predict the potential risk induced by the nanomaterial’s exposure on human health. We therefore decided to employ zebrafish as a vertebrate model to investigate the possible effects induced by benzoic acid functionalized CNOs (benz-CNOs, Fig. 1a left) and fluorescent boron dipyrromethene tagged CNOs (BODIPY-CNOs, Fig. 1a right). Different toxicological end-points such as the incidence of malformations, spontaneous movements and hatching rate/time disturbance are assessed during the zebrafish’s development. Moreover, the fluorescent BODIPY-CNOs biodistribution in zebrafish at completed organogenesis is studied using inverted selective plane illumination microscopy in order to probe their in vivo internalization inside the zebrafish larvae.


Biocompatibility and biodistribution of functionalized carbon nano-onions (f-CNOs) in a vertebrate model
(a) Schematic representation of benz-CNOs (left) and BODIPY-CNOs (right); Blue, nitrogen atom (N); pink, boron atom (B); green, fluorine atom (F). (b) HR-TEM image of BODIPY-CNOs.
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Related In: Results  -  Collection

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f1: (a) Schematic representation of benz-CNOs (left) and BODIPY-CNOs (right); Blue, nitrogen atom (N); pink, boron atom (B); green, fluorine atom (F). (b) HR-TEM image of BODIPY-CNOs.
Mentions: For any nanomaterial utilized in an application that might lead to a release to the environment or to the exposure of living beings, in particular humans, an accurate risk assessment as well as a toxicological screening is highly needed2728. The aquatic environment is of high importance since any contamination of water might lead to a wide distribution of the contaminant and thus to major pollution. All the biological in vitro studies investigating the effects of small CNOs (diameter of approx. 5 nm) with different surface functionalization on a variety of different cell cultures describe CNOs as a highly biocompatible nanomaterial. The effect of large CNOs (diameter of approx. 30 nm) on the immune system indicates that the cell response is highly dependent on the structure29. The inflammatory potential of small f-CNOs on immortalized bone-marrow-derived mouse macrophages and mouse bone-marrow-derived dendritic cells is found to be negligible and significantly lower than the effects of similarly functionalized single wall carbon nanotubes12. A recent report comparing large CNOs (diameter of 50–100 nm) with multiwall carbon nanotubes corroborates these findings30. We recently showed that small functionalized CNOs have no significant adverse effects on three weeks old freshwater polyp Hydra vulgaris31. The results reported on this very simple and basal animal, in addition to in vitro studies, suggest that CNO is a biocompatible and safe nanomaterial, but are not sufficient to rule out possible risks of CNOs exposure and release. Additional long-term toxicity studies of CNOs on complex organisms, as well as the fate of CNOs in biological systems, are strongly needed. Zebrafish represents an emerging and excellent model organism, mainly due to the fact that it has a remarkable similarity in the molecular signalling processes, cellular structure, anatomy and physiology to other higher order vertebrates323334353637. Moreover, zebrafish embryos are ideal for high-throughput screening due to their external development, optical transparency, and short breeding cycle3839. Zebrafish development represents a valuable tool to assess the in vivo toxicity and biocompatibility of drugs, chemicals and nanomaterials, with a focus on the developmental effects, and to obtain toxicity information at the whole animal level404142434445. Several approaches using zebrafish embryos or larvae as an animal model have been developed in recent years to assess embryonic effects of chemicals, drugs and nanomaterials464748, in order to predict the potential risk induced by the nanomaterial’s exposure on human health. We therefore decided to employ zebrafish as a vertebrate model to investigate the possible effects induced by benzoic acid functionalized CNOs (benz-CNOs, Fig. 1a left) and fluorescent boron dipyrromethene tagged CNOs (BODIPY-CNOs, Fig. 1a right). Different toxicological end-points such as the incidence of malformations, spontaneous movements and hatching rate/time disturbance are assessed during the zebrafish’s development. Moreover, the fluorescent BODIPY-CNOs biodistribution in zebrafish at completed organogenesis is studied using inverted selective plane illumination microscopy in order to probe their in vivo internalization inside the zebrafish larvae.

View Article: PubMed Central - PubMed

ABSTRACT

Functionalized carbon nano-onions (f-CNOs) are of great interest as platforms for imaging, diagnostic and therapeutic applications due to their high cellular uptake and low cytotoxicity. To date, the toxicological effects of f-CNOs on vertebrates have not been reported. In this study, the possible biological impact of f-CNOs on zebrafish during development is investigated, evaluating different toxicity end-points such as the survival rate, hatching rate, and heart beat rate. Furthermore, a bio-distribution study of boron dipyrromethene (BODIPY) functionalized CNOs in zebrafish larvae is performed by utilizing inverted selective plane illumination microscopy (iSPIM), due to its intrinsic capability of allowing for fast 3D imaging. Our in vivo findings indicate that f-CNOs exhibit no toxicity, good biocompatibility (in the concentration range of 5–100 μg mL−1) and a homogenous biodistribution in zebrafish larvae.

No MeSH data available.