SPIONs are well developed for biomedical applications as a consequence of their easy and reproducible production. They are in fact made from one of the most abundant metals present in metabolism: iron. Despite the fact that iron could induce ROS generation in cells [ 15 ], working with this element clearly decreases the potential toxicity compared to other metal oxide nanoparticles during dissolution processes in vitro or in vivo [ 15 ].
The Biogenesis of Cellular Organelles
Naked SPIONs tend to sediment and can precipitate at physiological conditions, leading to a severe toxicological hazard [ 16 ]. It is imperative to modify the surface of these nanoparticles to avoid any aggregation and the resultant risk of toxicity. Polyethylene glycol PEG is a polymer commonly used to increase the biocompatibility of the SPIONs as well as their stealthiness for specific targeting [ 17 ].
Many tests are measuring the evolution of absorbance and the nanomaterials are influencing the final value. It is then very important in order to avoid false positive or negative results, to carefully setup the experiments and the control to correct the absorbance [ 21 ].
As demonstrated in many studies, the addition of a biocompatible polymer layer on the SPION surface significantly improves their biocompatibility, which is a crucial step for the biological interactions targeted. First of all, the magnetic properties of SPIONs are very interesting to increase the cellular uptake rate of these nanoparticles with a magnet [ 22 ] and then to improve the labeling of cells for biomedical applications [ 23 , 24 ].
The concentration and the charge play a significant role in the cellular internalization [ 25 ]. For instance, negatively charged fluorescently labeled SPIONs have a higher internalization in prostate-cancer PC-3 cell line as observed via confocal microscopy or flow cytometry, in comparison to positively charged SPIONs [ 26 ]. Thus, the influence of the chemical coating is an important factor, however it seems that the nature of the medium used is much more critical. Regarding their biodistribution, SPIONs usually show accumulation in the liver and spleen [ 1 , 27 ].
They do not show any dramatic toxicity and have interesting cellular and in vivo interactions, making them extremely attractive as theranostic agents [ 29 ]. The cytotoxicity of titanate nanotubes made by hydrothermal treatment has been assessed in H human lung tumor cells [ 30 ], cardiomyocytes [ 31 ], SNB19 and UMG glioblastomas [ 32 ], Caco-2 cells [ 33 ], as well as 22Rv1 prostate cancer cells [ 8 ].
Interestingly, the degree of ion exchange via acid treatment, which partly or entirely substitutes sodium cations by hydrogen cations, is a key parameter that drives the cytotoxicity of titanate nanowires [ 30 ]. As described in the next section, in vivo studies have shown that TiONts acts like an anchor in the tumor, which prevents drug from leaching out of the cancerous cells, and as a result, the loss in drug potency was not detrimental in this specific case [ 8 ].
Indeed, an important intrinsic feature of these metal oxide nanotubes is their ability to potentiate gamma radiation effect on cells, making them an interesting candidate for combinatorial therapies on ongoing preclinical investigations i. This chapter mainly focuses on metal oxide nanoparticles, however, beyond the surface chemistry of such materials, one of the key parameters to consider while studying nanoparticle interaction with cells and tissues is their shape.
Indeed, due to their needle-like morphology, bare TiONts are internalized in cells not only by endocytosis, but also by diffusion across the plasma membrane, as observed by TEM analysis for cardiomyocytes [ 31 ], SNB19 and UMG glioblastoma cell lines [ 32 ]. Nanotubes display a significant higher specific surface compared to their spherical counterparts [ 2 ] and this potentially modifies their degree of interaction with plasma proteins and cells.
Our group has observed that even by incubating 4 times more spherical TiO 2 than TiONts with cardiomyocyte cells to account for the difference in specific surface values, TiONts were internalized in much more cells than spherical TiO 2 [ 31 ].
De novo peroxisome biogenesis revisited
Cell penetration via diffusion, along with their increased specific area, potentially makes them an excellent candidate as a new nanomedicine platform after careful assessment of their cytotoxicity in each targeted cell model. Nanotubes display unique behavior regarding their interaction with and internalization within cells, as well as distribution to tissues, compared to spherical nanoparticles. Indeed, the shape is a critical parameter governing circulation time and biodistribution for the same material.
For example, the circulation time for tubular micelles in mice is 10 times longer than the one of spherical micelles [ 35 ]. Interestingly, single walled carbon nanotubes have been demonstrated to be uptaken in the bloodstream by a subset of monocytes that subsequently deliver them to the tumor [ 38 ]. Nanoparticles passively accumulate in tumor by enhanced permeability and retention effect EPR effect , due to the poorly formed vasculature supporting the malignant cells, in combination with reduced clearance secondary to defective lymphatic drainage at site.
While passive targeted delivery to tumor is estimated to deliver only a small fraction of the injected dose utilizing spherical nanoparticles, nanotubes are capable of reaching significantly greater accumulation than their spherical counterparts and also display greater surface area that potentially leads to greater effect [ 39 ]. B Abundance of plasma proteins found at the surface of SPIONs after incubation with rat serum in vitro versus in vivo adapted with permission from .
Understanding the in vitro and in vivo behavior of nanoparticles is one of the main objectives of current studies. It seems too simplistic today to draw conclusions about their behavior without taking into account the environment, especially the proteins present in the systems studied [ 40 ]. Nowadays, it is accepted that once nanoparticles are incubated in biological fluids such as blood, they will be covered by proteins [ 41 ]. Not only do these proteins interact with the chemical coatings of materials, but they mostly also modulate their biological fate [ 28 , 42 ].
The nature of the coating, including resulting charge, surface chemistry and particle hydrodynamic size, influences the adsorption of proteins on the surface of nanoparticles: the protein corona [ 43 , 44 ]. For example, we demonstrated that bare silica beads covered by either a gold or a titanium oxide layer have different preferential binding to proteins [ 43 ]. We also showed there were important differences between in vitro and in vivo protein coronas [ 28 ].
Literature regarding the protein corona of TiONts is very limited at present and our group aims to elucidate key aspects of the topic in the years to come. Interestingly, titanate nanotubes bind significantly less plasma proteins than spherical TiO 2 Degussa P25 [ 46 ], even though they display a greater specific surface [ 2 ]. These proteins include albumin, Ig heavy chain mu , Ig light chain, fibrinogen alpha, beta and gamma chains and complement C3. The coatings of the nanoparticles influence the nature of their protein corona.
The medium used is also important for the interactions between materials and proteins [ 47 ]. Thus, taking into account not only the physicochemical properties but also the biological environment, it is essential to understand cellular uptake and biodistribution of nanoparticles in order to better control their toxicological risks. Organelles mitochondria, peroxisome, lysosome, endoplasmic reticulum, and Golgi apparatus are integral parts of the cells, essential for the its proper functioning. Their dysfunctions can lead to serious consequences.
For instance, mitochondrial alterations can go as far as to activate apoptosis [ 48 ], peroxisomal dysfunction affect the mitochondria, subsequently leading to oxidative stress and cell death [ 49 , 50 ], alterations of the lysosome may have consequences on the induction of autophagy and apoptosis [ 51 ], endoplasmic reticulum damages can lead to reticulum stress which can trigger different forms of cell death in extreme cases [ 52 ], and Golgi apparatus dysfunctions can disturb post-translational modifications and vesicular transport [ 53 ].
The incidence of the cytotoxicity of nanoparticles is often addressed in generalized terms such as induction of cell death, oxidative stress stimulation, inflammation activation and genotoxicity. The impact of nanoparticles on cell organelles is less known and must be taken into consideration as organelle dysfunctions affect general health in unexpected ways.
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As regards the peroxisome, whose dysfunctions can lead to severe neurodegenerative damage [ 54 ], there are currently no data on the effects of nanoparticles on this organelle. It is therefore essential to understand the interaction of nanoparticles with cell organelles in terms of distribution and impact on their biogenesis and biological activities.
This not only helps to prevent or optimize the toxic effects of nanoparticles depending on the intended purpose cytoprotection or cell death induction , but also to use them specifically in nanomedicine without side effects.
The interaction of nanoparticles with the mitochondria as well as the other organelles must be approached with the consideration that they are either the consequence of targeted interactions with specifically dedicated functionalized nanoparticles, or a random direct or indirect interaction which leads to unwanted side effects. This second aspect must be systematically taken into consideration, and integrated into a cytotoxic screening procedure which will permit to specify the biological activity of nanoparticles at the mitochondrial level.
In order to understand the toxicological interactions of nanoparticles on biogenesis and mitochondrial metabolism, it is necessary to specify whether they interact physically with the mitochondria and accumulate at specific locations such as external membrane, mitochondrial space, internal membrane and cristae. In this context, it has been shown that Gadolinium oxide Gd 2 O 3 nanoparticles, which have a range of biomedical uses, induce mitochondrial apoptosis by acting on Bcl-2 and Bax [ 55 ].
Similarly, silver nanoparticles impair mitochondrial activity and decrease cell viability [ 56 ]. Nanoparticles interact with mitochondria in different manner, based on their physicochemical nature. Since numerous types of nanoparticles are able to induce mitochondrial dysfunctions, which can have dramatic consequences on human health after chronic or acute exposures, a systematic evaluation of the impact of nanoparticles on the mitochondria is required.
Interaction of ZnO nanoparticles with murine microglial BV2 cells.
The ZnO nanoparticles exposure induced dose-dependent increase in transmitochondrial membrane potential and loss of lysosomal membrane integrity as revealed by flow cytometry analysis using fluorescent probes DiOC6 3 and propidium iodide respectively. Peroxisome has emerged as a key regulator in overall cellular lipid and reactive oxygen species metabolism. In mammals, these organelles have been recognized as important hubs in redox-, lipid-, inflammatory-, and innate immune-signaling networks. Peroxisomal dysfunctions are associated with important brain diseases [ 54 ].
To exert its activities, the peroxisome must interact both functionally and physically with other cell organelles, mainly mitochondria and endoplasmic reticulum [ 59 , 60 ]. It seems therefore important to precise the effects of nanoparticles on peroxisome. Nevertheless, no data are available concerning the impact of nanoparticles on this organelle.
Endocytosis is the major uptake mechanism of particles by cells [ 62 ]. The nanoparticles entrapped in endosomes are eventually degraded by specific enzymes present in phagolysosomes, as the endosomes fuse with lysosomes. The function of lysosomes is to break down molecules and dispose unwanted materials [ 63 ]. This phenomenon can also limit the delivery of therapeutic nanoparticles to the intracellular target site. Nanoparticles depending on its physicochemical nature can alter the function of lysosome and subsequently favor the activation or the inhibition of autophagy [ 64 , 65 , 66 ].
As the lysosomal pathway may have beneficial or detrimental effects on cell activity, a panel of assays is required to define the influence of nanoparticles on this organelle and its potential consequences in major diseases metabolic diseases, cancer and neurodegenerative diseases. Currently limited data are available on the impact of nanoparticles on endoplasmic reticulum and Golgi apparatus.
It has been reported that silica nanoparticles accumulate in the endoplasmic reticulum and triggers autophagy [ 67 ]. On the other hand, the intracellular accumulation of gold nanoparticles leads to inhibition of macropinocytosis and reduction of endoplasmic reticulum stress [ 68 ].
Thus, it appears that nanoparticles can have different effects on the endoplasmic reticulum. Consequently, their effects on this organelle must not be neglected. There is evidence that some nanoparticles can be taken up by the Golgi apparatus for further processing; however, no additional information are available on the influence of nanoparticles on the activity of the Golgi apparatus [ 69 , 70 ]. Among the most appropriated techniques available in nanotoxicology, observation of cells and tissues by TEM is well suited. This method permits quantitative and qualitative evaluation of modifications at the organelle level which are not easily detected with antigenic and functional changes.
Various methods of flow cytometry with appropriate probes are also of interest to define the impact of nanoparticles on the biogenesis and activities of the organelles. These methods make it possible to identify specific molecular targets and study the effects of nanoparticles on signaling pathways. The development of chip-based single-cell analysis is also of great interest for nanotoxicity assessment [ 71 ]. Overall, the beneficial or detrimental effects of nanoparticles on the organelles are difficult to predict. Systemic evaluation of nanoparticle interaction with organelles using simple techniques will help to minimize, if not to subdue, the biological risk associated with nanoparticles on human health, as well as with the environment.
Due to the increase of nanotechnologies in an expanding range of applications in industrial and biomedical purposes, those new materials require ecotoxicological, biosafety and biocompatibility evaluation.
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While nanotoxicity can be rapidly assessed in vitro , results obtained do not reflect complex processes that happen in full organisms and ecosystems. Various factors must indeed be taken into account, such as the route of administration i.