University of Groningen
Increasing the versatility of an ex vivo model in nanosafety studies and fibrosis
Bartucci, Roberta
DOI:
10.33612/diss.119127385
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Publication date: 2020
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Bartucci, R. (2020). Increasing the versatility of an ex vivo model in nanosafety studies and fibrosis. University of Groningen. https://doi.org/10.33612/diss.119127385
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Chapter
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NANOTECHNOLOGY IN HISTORY
The word nano-technology was coined and used for the first time by the Japanese professor Norio Taniguchi in a 1974 paper to describe semi-conductor processes at nano level[1]. However, it was the American engineer K. Eric Drexler in the 1970s and 1980s who was to be credited for the development of molecular nanotechnology (MNT), which led to the manufacturing of machineries at nano level[2]. One of the first con-tributors to the field was the Austrian-German chemist Richard Adolf Zsigmondy[3]. He studied colloids, which are chemical mixtures where particles of a substance are dispersed in another substance, including colloidal gold and other nanomaterials. For his research on colloids, Zsigmondy received the Nobel Prize in Chemistry in 1925.
Even though the term was coined only in the 20th century, the
appli-cation of nanotechnology started much earlier, for example in making steel and in paintings, which were in use centuries before the field was formally defined. Indeed, the use of nanomaterials started much earlier. One example is the lycurgus cup[4], also known as “roman nanotech-nology” and one of the greatest examples of nanotechnology in the ancient world. When the cup is lit from inside it will be red, while when lit from the outside it is green. This is due to the presence of nanopar-ticles, such as silver, gold, and copper, up to 100 nm in size, which are dispersed in the glass matrix[5]. Only after 1990, when the analytic tools were available, scientists were able to unravel the mystery of the lycurgus cup. It was discovered that the red color on the inside was due to absorption of light (roughly around 520 nm) by gold particles and the green because of light scattering by colloidal dispersions of silver particles with size >40 nm. The development of more sophisticated instruments, e.g. the atomic force microscope, and the availability of more powerful computers were essential in providing a better insight into nanomaterials and their properties[6].
Nanotechnology and its applications
Nanotechnology can be defined as the study and control of materials with nanoscale dimension, with 1 nanometer corresponding to 10-9 of
a meter in the metric system[7]. The prefix nano- can be used as a unit in relation to time, volume, or length. In the nanotechnology field, a nanomaterial is commonly defined as an object with a size in the range of 1–100 nm[8]. However, posing a limit to 100 nm does exclude a quite significant number of materials and devices, still behaving as nano-sized objects.
Indeed, the classification of nanomaterials is still debated in the field and it is not as easy as it may appear: as an example, are nanomaterials including also natural nano-sized objects or should they include only man-made nanomaterials? The opinion seems divided into research-ers that classify nanomaterials as all the objects smaller than 100 nm, regardless their origin (natural, incidental or engineered), and other currents of thought that refuse to include natural nano-sized objects into this definition. Regardless of this dispute, in most of the cases nanomaterials are usually classified based on intrinsic and extrinsic properties, with properties such as size, shape, morphology, mass and chemical composition belonging to the first category, and stability, surface charge, purity and corrosion belonging to the second[9].
Despite the small size of nanomaterials, nanotechnology applica-tions are many and their number is continuously growing. The use of nanomaterials can help to considerably improve, even revolutionize, Figure 1. Applications of nanoparticles. Reproduced from Current
Bionanotechnol-ogy, 2016[10] with permission. The scheme illustrates the many different applications of nanotechnology in very diverse fields.
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many technology and industry sectors, including not just electronics, but all sorts of areas of modern life, making nanotechnology the new technological revolution of our century (Figure 1)[10].
Here, a few examples of potential applications of nanomaterials are mentioned:
• for new renewable energy sources, for instance in solar panels to facilitate higher energy absorption or in lithium ion batteries to improve their performance[11];
• to detect pollutants in the air or in water in sensors, and potentially also to remove them, for instance for water remediation
• in electronics to replace high density memory chips with novel materials such as carbon nanotubes and overall to make electronic and IT devices smaller and more efficient;
• in food, where nanomaterials are used as additives to improve and affect some food properties, and in food packaging to im-prove them – for instance - in terms of durability and resistance to bacteria[12,13] ;
• in paints, to change properties of surfaces;
• in textiles, for instance to produce antimicrobial, water-repellent, anti-statics and thermal clothes[14];
• in cosmetics, for instance in as sunscreens to block UV-A and UV-B rays[15];
• to make materials with better mechanical properties, for instance for sport equipment and other applications;
• in nanomedicine as drug delivery system, and as imaging agents for sensing and diagnosis[16].
To summarize, nanotechnologies are used nowadays in numerous products and for many different industrial applications, they are pres-ent in our daily life and it has become very difficult to imagine a future without their use.
Nanomedicine and biological properties of nanomaterials
Many sectors have benefited from nanotechnology and the biomedical field, e.g. nanomedicine, is for sure one of them. Nanomedicines com-bine chemical and mechanical properties of nanomaterials to prepare new drugs that may reach their intended target with higher efficiency than standard drugs in traditional injections or pills. Nanomaterials are exploited for medical applications not only as novel drug delivery systems, but also for regenerative medicine, tissue engineering, for im-aging, and for diagnosis purposes[16–18]. In contrast to common small molecular drugs, which in many cases passively diffuse into the cells,
nano-sized objects are taken up by cells via energy-dependent processes and they are trafficked into specific sub-cellular compartments[19–21]. Thus nano-sized drug carriers open up the possibility of using cellular processes and pathways for targeting drugs into specific locations.
Indeed, one of the major goals of nanomedicine is to deliver better drugs for the treatment of various diseases, including among others cancer[17,22,23], fibrosis[24], Alzheimer[25,26], and to fight anti-microbial resistance[27]. Since the approval of the first nanomedicine, Doxil, liposomal doxorubicine, in 1995, roughly 50 nano formulations are currently available for clinical use (Figure 3). Among them, poly-mers (e.g. Plegridy[28,29]), liposomes (e.g. Doxil[30], Marqibo[31]), nanocrystals (e.g. EquivaBone[32]), micelles (e.g. Estrasorb[33]), inor-ganic nanoparticles (e.g. Dexferrum[18]) and protein-bound nanopar-ticles (e.g. Abraxane[34]) are some of the nano-drugs currently avail-able[35] (Figure 2).
But what makes nanomaterials so special and with such big potential for the biomedical field? Materials acquire unique properties once they are manufactured at the nanoscale[36]. In fact, material properties at the nanoscale are completely different from the ones that the same material possesses as a bulk[37]. One can simply think that using na-noscale dimensions means to move closer to the atomic or molecular scales. Atoms can be assembled in many ways, and both the chemistry and the geometric arrangement of atoms can influence the proper-ties of a material. Interatomic interactions and factors that are quite Figure 2. Types of nanoparticles in drug delivery already approved for clinical use. Reproduced from Pharmacy and Therapeutic, 2017[35] with permission.
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unimportant at macro-scale become dominant when materials reach the nanoscale, and many parameters change from those of the bulk material[36]. For example at the macro-scale, gold has certain char-acteristics that are not present at the nanoscale[38]. Additionally, the increase in surface area to-volume ratio is inversely proportional to the radius of a spherical particle, and when the same total volume of a ma-terial is made of nanoparticles, it comes as no surprise that the available surface area increases enormously. Altogether, when materials reach the nanoscale, they become more reactive than their bulk towards the surrounding environment. Additionally, in biological context, thanks to their size, nano-sized materials can easily enter cells[39], distribute within organisms. This is why potentially nano-sized materials can help drugs to reach their intended target. Despite the small size, at cell level, as mentioned, nanoparticles are too big to simply diffuse through the cell membrane, like common molecular drugs, but in-stead they employ endocytic pathways to enter the cells. The capacity to enter cells and distribute within organisms makes nanoparticles unique for biomedical applications, however, many factors still limit the development of nanomedicines, e.g. accumulation in non-desired organs which may lead to toxicity[40]. Indeed, achieving preferential distribution within the body to a specific target is still one of the biggest challenges in nanomedicine[41]. Big efforts are focused in reducing nanomaterial accumulation in off-target sites and increasing the re-tention of nanomedicines at their target[42,43]. Nevertheless, even though there is still room for improvement, the successes achieved so far in nanomedicine clearly show the potential of nanotechnology for biomedical applications.
Nanosafety and challenges in the study of potential impact of nanoparticles
As discussed in the previous section, the capacity of nanomaterials to distribute within organisms and enter cells opens up tremendous opportunities for their application in nanomedicine. However, at the same time it also raises concerns on potential risks for human health and environment associated to the unintended exposure to nanotechnologies.
Nanosafety is a scientific discipline, which studies the potential hazards of nanomaterials and their interference with biological sys-tems[44,45]. Understanding how nanomaterials are processed by cells and their impact at cell and organism levels may help to ensure the development of nanotechnologies with safer design and exclude
potential toxic effects due to intentional or unintentional exposure to nano-sized objects.
It is important to highlight that nanoparticle-induced toxic effects may also be the result of the modifications nanomaterials encounter in their lifecycle. Indeed, different factors may physically and/or chemi-cally transform nanomaterials[46] and, in order to reach solid conclu-sions on nanosafety, these modifications should be taken into account. Size, shape, purity, stability of nanoparticles in realistic exposure conditions are all factors that may affect nanoparticle impact on living organisms. At the same time, it has also emerged that scientific out-comes need to be carefully interpreted to exclude artifacts which may be created during laboratory studies if nanomaterials are not carefully handled[47,48]. For instance, monodispersed nanoparticles exposed to cell cultures may induce a different impact than aged or aggregated nanoparticles[49,50]. Thus, it is important to determine whether any observed toxic effect may be caused by the exposure to nanoparticles or may be due to aggregation and/or ageing of the dispersion. Similar considerations are in place if residual contaminants from the synthesis are still present or when metal nanoparticles are used. In this case, toxic effects on cells may be caused by the contaminants, rather than by the nanoparticles, or by the release of toxic metal ions[51–54].
Another aspect that must be considered is the different identity and therefore behavior that nanomaterials have on cells and organisms Figure 3. Synthetic identity versus biological identity of nanoparticles.
Repro-duced from http://joogroup.com/research[56] with permission. Nanomaterials are modified by the biological environment in which they are applied and acquire a new biological identity.
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once in contact with biological environments. The synthetic identity of nanoparticles differs significantly from their acquired biological identity (Figure 3)[55].
In fact it is nowadays established that the formation of the so-called corona, a layer of biomolecules adsorbed from the environment on the surface of the nanomaterial, changes material properties consid-erably[57,58]. The corona is formed by different biomolecules, pres-ent in serum, blood and any other fluid that the nanomaterial may encounter prior to contact with cells and organisms. Its formation and composition are highly dependent on the route of administration and/ or exposure[59]. The impact of nanomaterials at cell and organism level is strongly affected by corona formation[55,57,60]. Thus, nano-materials need to be tested in conditions allowing corona formation and – ideally – using biological fluids relevant for the cell or exposure route under study.
Biodistribution of nanomaterials
Nanomaterials are everywhere and humans can be exposed to them via external (air, water, food, etc.) and/or internal (surgical implant, drug delivery systems, diagnosis, etc.) sources[61]. Inhalation, ingestion or skin absorption of nanomaterials are examples of external sources, while direct exposure only happens when cells and tissues are directly exposed to the nanomaterials in nanomedicine applications (Figure 4). Primary effects of nanomaterials may occur at the exposure site, how-ever secondary effects may arise if the nanomaterials are translocated to other organs (as illustrated in Figure 4).
Nanomaterial distribution is affected by the physicochemical prop-erties of nano-objects. For instance, small nanoparticles in the size range of 1 to 5 nm are known to reach the kidneys and have a higher rate of renal clearance in contrast to bigger nanoparticles, that tend to accumulate in liver, lungs, brain, spleen, heart and the gastrointestinal tract[62–64].
The distribution of nanomaterials depends also on their different geometries: for example it has been shown that elongated polymer micelles have long-circulating lifetimes compared to spherical coun-terparts, and under fluid flow conditions, spherical nanomaterials are taken up by cells more readily than elongated particles at higher aspect ratio, namely nanoparticles with a length many times that of their width[65]. On top of size and shape, charge is another important factor in the fate of nanoparticles. Macrophages in lungs, liver and spleen seem to preferentially take up positively charged nanoparticles
rather than negatively charged and neutral nanoparticles and it has been shown that negatively charged nanoparticles and neutral ones have longer circulation lifetime[66].
Additionally, nanomaterials can be dissolved, decomposed, bio-trans-formed and/or completely degraded, depending on their composition, and these modifications affect their final fate as well as impact in vivo (Figure 5).
If nanomaterials are biodegradable, they may reduce in size and even-tually may be excreted in the urine via renal filtration[67]. However, nanoparticles that are non-biodegradable and larger than 5 nmmay be retained long-term in the liver, which is usually the organ where the highest accumulation is observed. Within the liver, nanoparticles are accumulated mainly by Kupffer cells, resident liver macrophages, followed by endothelial cells and hepatocytes[67]. If nanoparticles are not sequestered by Kupffer cells, they may undergo hepatobiliary elimination[67].
Next to the liver, smaller amounts of nanoparticles usually also dis-tribute into most other organs in the body. Within all organs to which they distribute, perhaps not surprisingly, a higher accumulation by cells of the immune system is commonly observed[66,68]. Generally, Figure 4. Route, potential accumulation and effects of nanomaterials.
Repro-duced from Dovepress, 2016[61] with permission. Nanoparticle exposure can occur through inhalation, ingestion, and via dermal exposure. Exposure can occur via internal sources, too in the case of nanomedicine applications. Nanoparticles can lead to pri-mary effects resulting from direct nanoparticle contact with cells and this may include toxicity, oxidative stress, DNA damage, and inflammation. However, nanoparticles may also translocate through tissue barriers into the blood, where they can circulate and eventually deposit in other organs, such as liver, spleen, or kidneys, where they could generate secondary effects.
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cells of the immune system, part of the mononuclear phagocyte system (MPS)[66,69], play a key role in eliminating xenobiotics from the body. The MPS includes many cells that are widely present in the human body, having in common the capacity of engulfing bacteria, apoptotic cells, viruses, and other foreign substances, including nanomaterials[70]. These cells are derived from precursor cells in the bone marrow, and in the blood they develop to monocytes and dendritic cells[71]. Most of them enter specific tissues and based on their location and phenotype, they take different names[72]: Kupffer cells in liver, alveolar macro-phages in lung, microglia in brain and so on[66].
Overall, if nanomaterials are not eliminated from the body, long term persistence may induce inflammation and toxic effects [73,74] in the organs in which the nanoparticles accumulate, and within these Figure 5. Decision-making flowchart to estimate nanoparticle fate in vivo sug-gested by Poon et al. Reproduced from ACS nano, 2019[67] with permission. Different
factors determine nanoparticle fate in vivo. The flowchart proposes a simple way to try to predict the final outcome and potential elimination or retention.
organs, in the resident phagocytic cells, usually shown to sequester higher amounts of materials.
Liver and nanoparticles
In vivo distribution studies have shown that the liver sequesters 30–99%
of nanomaterials from the bloodstream[75]. This organ consists of 60–80% of hepatocytes, which are specialized epithelial cells, and of non-parenchymal cells[76]. The non-parenchymal cells include Kupffer cells (resident liver macrophages), liver sinusoidal endothelial cells, hepatic stellate cells, biliary epithelial cells (cholangiocytes), resident immune cells (dendritic cells, natural killer cells and lymphocytes) and circulating blood cells that pass through the organ[77].
Kupffer cells make up 80–90% of the total body resident macrophage population and are the main responsible of the phagocytic activity in the liver[78] (Figure 6). In vivo distribution studies, in fact, showed that despite the high fraction of hepatocytes, nanoparticles are mainly taken up by the non-parenchymal cells, and in most cases by the Kupffer cells [79].
Kupffer cells are localized in the liver sinusoids, and constitute the first cellular barrier that nanoparticles encounter when the blood enters Figure 6. Scheme of nanoparticle uptake in liver and possible clearance via hepatobiliary elimination. Reproduced from Journal of controlled Release[75] with
permission. Nanoparticles enter the liver via the portal vein (1), then they enter the sinusoids (2) and are in contact with Kupffer cells (3). The nanoparticles that are not taken up by Kupffer cells may transcytose through the hepatocytes (4) and enter the bile ducts via bile canaliculi (5). Nanoparticles are then in the hepatic ducts (6) and they may collect inside the gallbladder (7) or they may enter the common bile duct (8), and be excreted into the duodenum (9) and finally eliminated in feces (10), after traversing all the entire gastrointestinal tract.
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the organ via the portal vein. Kupffer cells can recognize opsonized pro-teins, specific surface chemistries, and the protein corona that marks the nanoparticles, and internalize them through multiple scavenger receptors involved in different endocytic mechanisms[80,81]. Thus, clearly, Kupffer cells play a key role in nanoparticle uptake within the liver and they are known to accumulate a high amount of nanomate-rials. If nanoparticles are not exported or degraded, they may have long-term retention in these cells. Long and persistent accumulation of non-degradable materials may lead to inflammation and toxicity or subtle alterations of the functions of these specialized cells[79,82]. A relatively small amount of nanoparticles that reach the hepatocytes in the end can instead be eliminated via the abovementioned hepatobi-liary excretion.
For nanomedicine applications, sequestration of nanomaterials by the liver remains one of the barriers to drug delivery. Making nanomed-icines “bioinvisible” to the host to reduce macrophage recognition in
vivo might help to enhance their circulation time for improved delivery
and efficacy. Reducing protein adsorption for example with the use of polyethylene glycol (PEG) and other hydrophilic polymers commonly utilized for drug delivery[83] is also known to help prolonging plasma residence time of nanomedicines. Eventually, also these systems are recognized by the innate immune system, taken up and removed from circulation[84].
Overall, understanding more of the behavior of nanomaterials in the liver is needed both to improve nanomedicine success and to better prevent potential toxic effects due to unintended exposure to other nanoparticles accumulating in this organ.
Lungs and nanoparticles
Foreign materials, such as viruses, bacteria and nanomaterials, are present in the air we breathe, and if small enough, they can be in-haled[85]. The respiratory system, composed of almost 40 different cell types, is very complex, and it includes the trachea, bronchi, bronchioli and down to the gas exchange zone, the alveoli[86].
Depending on their size, particles can deposit in different regions of the lung and different clearance mechanisms are in action[87,88]. In general, it is suggested that microparticles (0.5–10 μm) are directly transported back upwards by mucociliary clearance, also called the mucociliary escalator[87]. Afterwards, mucus containing foreign sub-stances may be removed by coughing. In contrast nano-sized objects can deposit in the entire respiratory tract with high efficiency and
may reach the blood-air barrier by diffusion[88]. It is suggested that clearance of nanoparticles may occur by ciliated mucociliary cells of the upper airways (Figure 7)[87], while in the alveolar region, alveolar macrophages, together with epithelial cells (Type I and II), play an important role for clearance.
One of the classical hypotheses is that particles inhaled into the lung are taken up by alveolar macrophages, and trigger an inflammatory response within the lung[87,89]. There is, however, certain evidence that nanoparticles taken up by the lung can also end up in secondary organs such as the kidneys, liver, heart, brain and spleen[88,90,91]. Furthermore, it is well-known that geometry and charge are also im-portant characteristics to consider in relation to potential toxicity of nanomaterials in the lungs. For instance, it is reported that fiber-like nanoparticles may induce effects similar to asbestos and that cationic Figure 7. Possible ways of clearance of nanomaterials in the lungs. Reproduced
from environmental health perspectives, 2017[87], with permission. Nanomaterials arriving in the alveolar space can be cleared via mucociliary escalator, or after crossing the epithelium they may reach the lymphatic vessels, or translocate in blood capillaries and reach the systemic circulation. Additionally, the alveolar macrophages may take them up and translocate to the lymphatic vessels or to the blood circulation. Further-more, it has been suggested that another route of clearance from the lung may be via re-entrance back onto the alveolar epithelial surface (via an unknown mechanism) for long-term macrophage-mediated transport toward ciliated airways and the larynx.
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silica nanoparticles in the lung can lead to chronic injury and inflam-mation, while particles with polar or anionic surfaces tend to migrate to the mediastinal lymph nodes[92].
Surface charge and surface structures influence indeed the biokinetics of nanoparticles. They affect the interactions of nanoparticles with pro-teins and cellular components, and thereby the mechanisms of particle translocation and accumulation in extra-pulmonary organs[93,94]. Understanding the behavior and potential toxicity of inhaled nanopar-ticles may help to gain more information also on nanoparticle effects in secondary organs.
Kidneys and nanoparticles
Nanoparticles small in size (<5 nm) are mainly excreted via renal fil-tration[67,95,96]. The kidneys represent, together with the MPS, the second major route of nanoparticle clearance[97].
This organ includes three different regions: the renal cortex, medulla and pelvis. The main functional unit is the nephron, placed in the cor-tex and medulla. The nephrons are composed of the renal corpuscle, proximal tubule, loop of Henle, distal tubule, collecting ducts and per-itubular capillaries. The main function of the organ, namely filtration, happens in the glomerular filtration membrane (GFM), composed by the glomerular basement membrane (GBM), endothelial cells, endo-thelial glycocalyx, and podocytes, supported by mesangial cells in the mesangial matrix[97,98]. The GFM and the mesangial cells play the main role in nanoparticle filtration. The properties of the nanoparticles again determine their fate: big nanoparticles (100 nm) cannot pass the membrane of endothelial cells, while nanoparticles between 100 and 6 nm cannot pass the GBM, while nanoparticles between 2 and 6 nm have a fast clearance. Instead, nanoparticles smaller than 2 nm can have slower clearance, because they are so small that they can be trapped by the glycocalyx, thereby reducing the filtration time. Not only the size, but the charge and the shape of nanoparticles can impact renal clear-ance. The endothelial glycocalyx, GBM and podocyte glycocalyx are negatively charged. Because of this, positively charged nanoparticles
may be cleared faster than neutral ones, followed by negatively charged
nanoparticles[97]. Charge density also matters, thus highly negatively charged nanoparticles are cleared faster than less negatively charged nanoparticles. The shape is another important factor that affects kid-ney filtration because of the shape of the GBM: it has been observed that, for instance, high aspect ratio nanoparticles with a width smaller than the kidney filtration threshold (smaller than 5.5 nm) can also be
filtrated through the kidney[99]. Overall, the physical properties of nanoparticles determine their interactions with different kidney com-partments and thereby affect the potential targeting of nanomedicines to this organ ,as well as nanoparticle clearance. A better understanding of the mechanisms of uptake and potential clearance of nanomaterials in the kidney and all other organs where they distribute remains es-sential both for the design of targeted nanomedicines and to address nanosafety.
NANOSAFETY, ACHIEVED GOALS AND OPEN
QUESTIONS
Whether nanomaterials are intended to be used for biomedical ap-plications or enter and distribute into organisms after unintentional exposure, it is highly necessary to investigate their potential toxicity. The main goal of the nanosafety community is to understand whether persistent exposure to nanomaterials and accumulation can lead to conditions of oxidative stress, inflammation and perhaps to the de-velopment of severe pathological conditions. In these last decades the field of nanosafety has significantly developed, and the continuous increase of the number of scientific papers on nanosafety reflects the huge efforts done so far by this scientific community[100]. As discussed above, it is now well established and proven that nanomate-rials can cross e.g. the lung and gastrointestinal tract, reach the blood stream, and consequently other secondary organs, such as spleen, liver, and kidneys. Although systemic effects have also been observed, com-parison of the many results and scientific papers in literature is often complicated due to (in most cases) the lack of standardized operating procedures adopted during laboratory experiments.
Many questions are still unanswered, due to several reasons, such as: • the lack of appropriate parameters to define nanomaterials and
proper nanomaterial classification;
• the lack of standardized methods for hazard assessments for in
vitro and in vivo studies;
• (sometimes) incorrect nanomaterial exposure in lab experiments that leads to non-realistic outcomes (for instance exposure of bare nanomaterials to cells in the absence of some biological fluid to allow corona formation);
• difficulties in connecting potential hazards and risks of nano- materials;
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• lack of accurate advanced in vitro models;
• discrepancies between in vitro and in vivo outcomes.
Thus, precision, standardization, reliability, translatability and re-producibility are all key factors that are needed for the progression of knowledge in the nanosafety field.
One of the major problems in testing engineered nanomaterials is to find reliable, cost- and time-effective advanced in vitro models that may substitute or reduce the need for animal testing[101]. While in
vivo studies are mostly focused on the accumulation of nanoparticles
at organ level, most in vitro studies are focused on a single cell type in culture. In these simpler models, the unique architecture and orga-nization of cells within the organ of interest are often not considered.
Next to this, given the continuously increasing number of nanoma-terials, the assessment of their impact in vivo can become cost-inten-sive and time-consuming. Moreover, outcomes from animal studies may poorly correlate to potential impact in humans, because of the physiological and biochemical dissimilarities across different spe-cies[102]. Given the high number of different cells present in human body (roughly 200 types), organized in different tissues and organs, it is really complicated to develop a single model to fully reproduce in vivo studies. Nevertheless, the combination of the most relevant in vitro models may help to capture essential features of in vivo studies to gain a better knowledge on behavior, impact and potential toxicity of nanomaterials.
To this aim, new in vitro systems such as 2D and 3D cell cultures, co-cultures, organoids, organs-on-a-chip are being developed, vali-dated and exploited as advanced in vitro models, supporting the prin-ciple of the 3 R’s - reduction, replacement and refinement of animal models[103–106]. Aiming to reducing the gap between results obtained on cell cultures and animal models, many studies using advanced in
vitro models have been published[101,103,106]. For instance,
mono-cultures and co-mono-cultures that mimic different part of the lungs are being used, the lungs being the organ that is most exposed unintendedly to nanomaterials. In order to mimic some key aspects of the lung, the combination of epithelial and endothelial cells with alveolar macro-phages in a transwell system is nowadays in use to study the potential impact of nanomaterials on this organ, including the use of air liquid interface cultures[106]. Lung organoids have also been developed and their use is increasing. However, despite the advantages and promising results reported, these and other similar models still lack one important factor that should not be neglected, which is the cellular complexity
of a real piece of tissue. Ex vivo models may be useful alternatives to fulfill this aspect.
Precision-Cut Tissue Slices
Precision-Cut Tissue Slices (PCTS) are a 3D ex vivo model, well es-tablished and mainly used for drug metabolism and toxicity stud-ies[107,108]. Moreover, PCTS are exploited to study onset and end-stage of diseases such as fibrosis[109–114], thus they can be used to investigate the role of proteins involved in this particular patholog-ical condition, e.g. osteoprotegerin[115] and in this thesis, vanin 1 ( Chapter 6).
Recently, tissue slices, especially when extracted from lungs, have been used as a valuable tool also in the field of nanosafety[43,116–120]. PCTS represent a promising alternative to simple cell cultures: a better
in vivo-like environment is resembled in a real piece of tissue of typically
5 mm diameter.
PCTS are fast to prepare with accurate reproducibility, as well as easy to handle[107]. Potentially any animal model can be used, although slices derived from rat and mouse are exploited most frequently. Im-portantly, they can be also prepared from human tissue using biopsies, surgical waste and non-transplantable organs[112,114,121,122]. The starting human material can derive from healthy as well as diseased tissue, broadening the spectrum of possible investigations even further. The possibility to conduct research in human tissue is one of the biggest advantages of this model to reduce the gap between results obtained using animal models and human data.
Tissue slices can be extracted from different organs such as liver, lung, kidney, intestine, and brain[107,111,113,122–124]. The cell architecture, cell ratio and conformation are maintained intact and represent the organ-specific environment in miniature. In fact, all cells, including more sensitive ones, such as macrophages, are kept embedded in their in vivo environment. Although using a piece of real tissue is a great advantage, the lifespan of precision-cut tissue slices is still relatively short (even though lung slices can be cultured up to two weeks), especially if compared to advanced models that are kept in culture for several weeks[125].
Protocols for extraction and preparation of PCTS are well established and allow obtaining slices of well-defined size with good control. After extraction of the organ of interest, cores are prepared using a drill or a biopsy puncher with a diameter of 5 mm, or in the case of murine kidneys the entire organ is used for further steps. The cores are then
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positioned in a slicer and the slicing procedure is performed with an automatic oscillation of the knife inside the machine. Slices with a thickness of roughly 200–250 μm are produced and are ready for fur-ther use (Figure 8).
Previous studies have shown that PCTS still display species-, organ- and pathology-specific differences in gene regulation[126–128].
Although so far PCTS have mainly been used to investigate toxicity of traditional drugs, some publications have shown that PCTS can also be a valuable tool for the assessment of nanomaterials, in terms of inflam-mation and toxicity. The first studies using PCTS with nanoparticles have been mainly focused on determining the impact of nanomaterials in lung slices[43,116,118,120,129], while fewer examples are available for liver[117], intestinal[130] and tumor slices[131]. Most of these studies, except two in which human tissue was used[130], focused on tissue slices prepared from animal models.
Some studies have also reported difficulties of using this model for nanosafety and nanomedicine approaches[101]. In fact, nanoparticles are taken up via energy-dependent processes[132], therefore they need to be in contact with living cells. Possible debris and dead cells on the surface of the slices following the cutting procedure may adsorb nanoparticles, thus they may constitute an obstacle for uptake. How-ever, protocols are optimized to reduce the presence of dead cells at Figure 8. Preparation and incubation of rat liver slices. Nature protocols, 2010[107],
with permission. Rat liver is extracted and with the use of a drill (a), cores with a di-ameter of 5 mm are prepared (b). The cores are then positioned in the core holder of a Krumdieck slicer (c–d) and slices with 15 mg wet weight are cut (e). Slices are made with equal thickness and shape (f). As last step, slices are transferred to 12-well plates using a spatula to avoid damaging the slices (g–h).
the slice borders by introducing a pre-incubation time for the tissue to fully recover after slice extraction prior to experiments.
Despite several examples using PCTS for nanosafety, a careful study of nanoparticle uptake and distribution on tissue slices using for instance fluorescence microscopy to image the full tissue has not been reported yet and may give important insights on the applicability of this model for nanosafety. Only in one study nanoparticle uptake in tissue slices was investigated by imaging using electron microscopy[117]. Addition-ally, enzymatic digestion of the tissue slices after nanoparticle exposure
ex vivo and further analysis on the recovered cells by flow cytometry
may give further insight on nanoparticle distribution within the tissue, as suggested by Park et al. who conducted a similar study in vivo[133]. A potential limit to the use of tissue slices for nanosafety studies is related to how nanomaterials are exposed to tissue slices. In vivo nanomaterials encounter specific cells, depending on the route of expo-sure or administration, whereas ex vivo they may enter in contact with whatever cell type is present in the first layer of the slice. In the case of liver slices, for instance, nanomaterials may enter preferentially in the hepatocytes, being the cells mainly populating this organ, as opposed to Kupffer cells, in which the highest uptake in vivo is usually observed[75].
In addition to this, when PCTS are used for studying nanomaterial be-havior, conditions suitable for both tissue maintenance and nanoparti-cle exposure need to be applied. For example, PCTS are usually cultured in serum-free medium[111,112], but for realistic exposure, nanopar-ticles have to be dispersed in medium containing a source of biological fluids. As abovementioned, it is well-known that the presence of bio-molecules adsorbing from the biological environment on the nanopar-ticles affects strongly their interactions with and impact on cells[55,57]. Therefore, to avoid artificial results due to unrealistic exposure of tissue to bare nanoparticles, biological fluids have to be included. Additional effects due to the ageing of nanoparticle dispersion, especially during long exposure experiments, must be also considered[134,135]. Know-ing that for many nanoparticles no degradation and/or export has been observed, it is indeed important to address whether long-term effects can be studied in this model, using appropriate conditions.
To summarize, appropriate approaches have to be developed, tested and validated when nanomaterial behavior is investigated ex vivo in PCTS, and only then an attempt can be made to address the potential use of PCTS for nanosafety. Several questions still need to be answered. For instance, how do results obtained on simple cell cultures in vitro translate to ex vivo results?
1
Intr
oduction
To which extent is it possible to translate ex vivo results observed in
vivo using PCTS?
Is it possible to observe nanomaterial uptake in PCTS and, if so, in which cell types?
Considering the relatively limited lifespan of PCTS, can long-term exposure experiments be performed on PCTS to test potential chronic effects of nanomaterials or to test inflammation status and/or initiation of some pathological conditions?
Answering these and similar questions will allow to understand whether tissue slices can be used to obtain meaningful information on the impact of nanomaterials.
The work presented in this thesis represents a first step towards this direction.
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AIM OF THE THESIS
Precision-cut tissue slices are an interesting ex vivo model already vali-dated and extensively used for toxicological studies, drug metabolism, and also as a model for early-stage and end-stage of fibrosis and to study the role of specific proteins during the development of fibrosis over time[1–4]. The maintained cellular complexity of the tissue and the possibility of preparing tissue slices from diverse species, including from human tissue, and different organs, are only few of the many advantages of using this model.
In this thesis, tissue slices have been exploited as an ex vivo model for nanosafety studies (PART I) (Figure 1) and to investigate the potential alteration of vanin 1, a protein involved in oxidative stress and inflam-mation[5], during the different stages of fibrosis (PART II).
PART I
Significant efforts within the nanosafety community are currently fo-cusing on the implementation of novel advanced models for in vitro testing. Ideal models should resemble the complexity of the in vivo environment and key features of the organs in which nanomaterials accumulate. Nanosafety is therefore attempting to exploit the use of Figure 1. Schematic summary of the studies conducted in PART I of this thesis.
