**3. Ex vivo models**

Using *in vitro* models for studying nanoparticle mediated toxicity comes with the set of challenges as discussed in the previous section. Animal models like rodents or zebrafish embryos are ideal alternatives for studying toxicity induced by NPs, however, there are various limitations including the cost of maintenance, biosafety etc. One also needs to follow appropriate ethical guidelines and have a moral compass to ensure whether inducing pain or sacrificing the animal can be avoided by alternate studies.

*Ex vivo* studies where tissue slices are cultured outside the host organism, experiments on fertilized eggs, organs on chip studies are more reliant than *in vitro* studies which are often based on monolayer culture of cells. The following section will briefly describe the prominent ex vivo models.

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### **3.1 Precision cut tissue slices**

In precision cut tissue slices, tissues are collected from a model organism or human biopsy samples for testing the effects of nanoparticle treatment. This approach provides a more comparable biological scenario rather than using immortalized or primary cells cultured in monolayer. As the organ tissues provide a better biological replica of the effect of nanoparticle in the biological system as a whole. Predominantly, this method has been used to study aerosol effects or nanocarriers developed for treating lung disorders [93–96]. Using liver cut slices to study hepatotoxicity is also common [97–100].

One of the major challenges of this method involves the penetration of nanocarriers into the organ slices. In a biological system, like our body, circulatory system ensures the uptake/delivery of nanocarriers. Without this in the organ slices which also has several physical barriers makes it difficult for the passive entry of NPs. As seen in some studies, the NPs are seen around the surface of the tissue slices [101]. Besides this, sometimes slicing a tissue induces inflammation around the cut region. This is unfavorable while studying toxicity effects. An inflamed tissue could introduce bias into the experimental outcomes. Inflammation could activate cells and induce necrosis or apoptosis which will interfere with toxicity results for nanoparticle testing.

#### **3.2 Organ-on-a-chip**

A 3D culture with multiple kinds of cell population sufficiently supported with extra cellular matrix and growth factors is better than monolayer culture in terms of nanotoxicology analysis. However, these kinds of system are considered to be static and does not mimic vascular perfusion or the sheer stress in biological systems. Recently, microfluidics have been integrated to 3D culture of different population to generate micro physiological systems called organ-on-a-chip. A very simplistic model of organ-on-a-chip will have a single type of cell lined over the microfluidic channel with a continuous flow of media. In advanced models like lung-on-a chip, blood brain barrier-on-a chip or blood retinal barrier-on-a-chip model, multiple types of cells are used with microfluidic channel mimicking biological processes like breathing, blinking [102–115] etc.

These devices employ different perfusion rates and with the kind of material used, introduce mechanical strains which mimics biological circulation events like breathing, heartbeat, peristalsis, blinking, twitching etc. By using transparent materials for channel construction, the device also allows live visualization. As compared to conventional *in vitro* techniques, this provides a better physiological parallel with intricate designs to replicate organ architecture. Recently, there have been studies to develop multiple organ on a chip interconnected devices which could essentially be called body-on-chip to develop a model of whole organism rather than studying the effects on individual organs [116]. With intricate designs to carefully recreate biological systems, this could to a larger extent eliminate animal based testing.

Fluorescently labeled PEGylated gold nanoparticles were tested on tumor on a chip model which provided better insights into the circulation, elimination and uptake of nanoparticles in tumor microenvironment [117, 118]. Similarly Organ-ona-Chip model are used in understanding the effects of shape of NP on its toxicity [119]. Understanding the effect of flow rates, especially while NP mediated targeted therapy for crossing BBB [120], the underlying changes in toxicity profile introduced by surface modifications of the particle [121] etc. are few of the recent advances in NP mediated research which has recently been revolutionized by Organ-on-a-Chip models. Specifically focusing on toxicity studies, lung-on-a-chip models have provided better insights into pulmonary toxicity of nanoparticles, specifically, TiO2, Silica NPs [108, 122] etc. Also, the effect of NPs on crossing placental membrane has been evaluated using Organ-on-a-Chip model. In one such model, placental membrane integrity and maternal immune cell response were negatively challenged by TiO2 NPs [123]. These models also enable the researchers to evaluate toxicity of NPs in both static and mobile conditions as opposed to static conditions in *in vitro* studies [124].

One major disadvantage of the devices is the use of precursors which adsorbs drugs while testing. This reduces the amount of drugs available for interacting with the cells of interest. The widely used material polydimethylsiloxane (PDMS) [125] is known to adsorb certain drugs which is a disadvantage to the application. Polysulphone based materials are considered to be an alternative for PDMS. However, they lack transparency and are difficult to tune the mechanical properties. If careful considerations are introduced while designing the architecture and appropriate precursor materials are chosen, organ-on-chip can be a lot desired application for nanoparticle toxicity analysis (**Figure 5**).

#### **3.3 Chick chorioallantoic membrane (CAM) assay**

Angiogenesis and neovascularization is a characteristic trait of multiple disorders including cancer and retinopathies. Preventing the formation of new leaky vasculature is thus critical in treating these disorders. NPs developed as potential therapeutic options thus need to be evaluated for their anti-angiogenic properties. Evaluating this on developing chick embryos is both cost and time effective other than the simplicity of the model [127–130]. Briefly, the assay is performed by making a hole in the eggshell and inserting a membrane which delivers the NPs of choice. At the end of study, the eggs are opened, and the blood vessels are quantified using bright field imaging. One of the major advantages of this method is its reproducibility and simplicity to conduct in small scale laboratories.

#### *3.3.1 Ex-vivo tumor models on CAM*

Cancer cells of human origin are transplanted to CAM that covers chicken embryo. After 3 days of transplantation, a tumor with host species features will be developed. The tumor features multiple cell type of human origin, with rich vasculature and extracellular matrix. In terms of tumor microenvironment and cell types, this model is a closer approximation to *in vivo* models compared to 3D organoid studies. Also, the tumor formation process is rapid and eliminates longer waiting period as compared to rodents. Also, immune compromised animals are costlier in contrast to chick embryos which poses naturally underdeveloped immune system in early developmental stages. Besides these advantages, the rich nutrients present in chick embryo encourages effective angiogenesis in the tumor model. This facilitates drug and nanoparticle testing and monitoring of reduction of micro blood vessels and other tumor characteristics (**Figure 6**).

#### *3.3.2 Anti-angiogenesis studies for retinopathies*

These are studies where implants are prepared and investigated on its ability to sustainably deliver therapeutic agents in ocular regions. Neovascularization and

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### **Figure 5.**

*The lung/liver-on-a-chip platform. (a) A photograph of the chip system comprising the pump main unit with four pump heads, the PEEK chip, and the reservoir plate. (b) a schematic view of the chip comprising four circuits is shown; each circuit includes two compartments to house the lung and liver tissues, respectively. The cross-section schemas of the plates show the path of the tubes and channels and the relative depth of each well. (c) a close-up view of the two compartments showing the groove pattern on the bottom of the wells. (d) Effect of chip materials on absorption of nicotine. A solution of 10 mM nicotine in phosphate-buffered saline (PBS) was kept in the wells of the chips made from PEEK or PDMS for 8 hours before it was collected. The concentrations of nicotine were then measured using liquid chromatography coupled to high-resolution accurate mass spectrometry. Nicotine concentrations remaining in solution are expressed as % relative to the stock solution. Data are presented as mean ± SEM. N = 3. PEEK: Polyetheretherketone; PDMS: Polydimethylsiloxane [126].*

resultant increased ocular pressure lead to a plethora of ocular disorders including diabetic retinopathy, macular degeneration etc. Nanoparticle mediated implants or delivery systems attempt to target pro-angiogenic markers in the eye. However, this needs to be carefully implemented with increased attention on not to induce any inflammation and reduction in angiogenesis.

**Figure 6.**

*Patient tumor sample transplanted on the CAM membrane. (A) Tumor formed by transplanting minced sample of ovarian cancer patient tumor. (B) Tumor is eliminated after intravenous injection of PMO-1 containing doxorubicin. (C) Chick embryo major organs look normal 3 days after injection [130].*

Briefly fertilized eggs will be candled to spot the air sac and blood vessels. A small hole of 1×1cm will be made into the egg shell at air sac region. Similarly, a cut is made near the vascular region without disturbing the CAM. Sucking out air from the air sac will distant the CAM from the egg membrane at the vascular region. Once this is achieved, a small incision is made at the CAM of vascular region where implants are introduced through sterilized filters. These filters containing the particle are then incubated over desired time frame. At the end of experiment duration, CAM is fixed and imaged to count the no of blood vessels compared to untreated controls (**Figure 7** represents multiple ways of performing CAM assay for angiogenesis studies).

For a simplistic and cost-effective model like CAM assay, most often the only disadvantages lies in the frequent contamination of samples. This can be eliminated with limited exposure of the opened CAM to outside air and by following stringent sterilization practices [132].

### **4. In vivo models**

Nanocarrier formulations are tested in in vivo models as pre-clinical studies to evaluate its feasibility to escalate to clinical trials and into commercial market at the end of pipeline. The maximum dosage of NPs which could be safely tolerated, the pharmacokinetics and elimination window of NPs from the tested organisms, the accumulation and effect of long-term exposure is analyzed during these experiments. Vertebrates and invertebrate groups of animals are used for these studies to scale up from simplistic biological networks to understanding the effects on complex In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### **Figure 7.**

*Representative images of chorioallantoic membrane (CAM) variants. (A) in-ovo setup by windowing method on day of incubation; (B) ex-ovo setup in a petri plate; (C) ex-ovo setup in a glass-vertical view; (D) ex-ovo setup in a glass-horizontal view; (E) ex-ovo setup on plastic cups, image taken by a camera and; (F) ex-ovo setup in plastic cups, image taken by a Chemidoc (charge-coupled device (CCD) camera) [131].*

organisms genetically closer to human beings. Good lab practices are stringently adhered to while conducting these studies with appropriate ethical standards.

#### **4.1 Invertebrates**

Invertebrate models often have a shorter lifespan which aids researchers in testing nanoparticle toxicities. Due to their shorter life cycle, it's feasible to understand and compare the effects of exposure of NPs in their developmental stages. It also benefits to conduct multiple rounds of testing within a shorter duration of time. The most established invertebrate model systems for nanoparticle toxicity include *Caenorhabditis elegans* and *D. melanogaster.*

#### *4.1.1 C. elegans*

*C. elegans* are nematodes which can grow up to 1 mm in size in its fully developed adult stage. They are often used for understanding nanotoxicity through oral uptake which is also the major form of nanotoxicity in human beings [133–138]. They pose around 70–80% of gene homology with humans and have around 70% of major signal transduction pathways conserved as compared to human beings. They are also transparent in nature allowing to visualize and track the accumulation of fluorescent labeled NPs. (**Figure 8**).

Major studies conducted on testing nanoparticle formulation and its effect on *C. elegans* has found that there have been significant changes in oxidative response, reproduction and lysosomal signaling after oral uptake of particles. For example, treatment with TiO2 NPs have reported to alter the expression levels of glutathione-stransferase gene. Exposure to NPs in *C. elegans* could also be through their vulval slit or opening. The nematodes being hermaphrodite, interaction of NPs at vulval site and spermathecae, where sperms are stored and oocyte fertilization happens, could provide preliminary results on how NPs affect reproduction in organisms. Interestingly, a recent study has reported that exposure of TiO2 NPs in *C. elegans* leads to decreased expression levels of pod-2 a gene known to have role in reproduction in the nematodes [133, 136]. Another study has reported the exposure of silver NPs leading to altered expression levels of proteases involved in lysosomal pathway related genes.

Generation of mutant strains of *C. elegans* has helped in understanding the effects of common particles like graphene oxide (GO), Silver, cadmium quantum dots and TiO2. Tracing of these NPs is often reliable and easy in *C. elegans* due to the transparency of its body. However, *C. elegans* lacks organs like a well-developed lung, kidney, heart etc. which makes it difficult to draw comparisons with higher order species. Also, it only has a 70% homology with human genome with some critical signaling pathways completely absent.

#### *4.1.2 D. melanogaster*

*D. melanogaster* or fruit fly is another model well used for oral toxicity of NPs. Like *C. elegans*, fruit fly poses different life development stages which makes it suitable to study the effect of NPs on different life stages. Effects of NPs in the gut cells, eye and wing development are often studied for testing the toxicity of NPs (**Figure 9**). Along with this, behavioral studies are also conducted where crawling speed and path is monitored [140, 141]. Immune pathway in fruit flies is well studied and they also display a similarity of autophagy related genes with human beings. This makes *D. melanogaster* an ideal model for studying immune response and rate of autophagy with respect to NPs like GO [142]. The effect of nanoparticle exposure to the organisms is also analyzed by studying the effects on reproduction. Offspring number, morphology, development life stages are analyzed to understand the same [140, 143–145].

The studies of nanotoxicity using fruit flies are limited to survival, developmental stages, eclosion rate, fertility and geotaxis performance analysis. Though it provides a significant addition in terms of understanding the toxicity as compared to *in vitro* studies, parallels cannot be drawn between human beings. One of the major challenges in using *D. melanogaster* as a model organism is the absence of an adaptive immunity in the organism. Other limitations include insufficient evidence of the cognitive capabilities of the organism especially affecting behavioral studies. Also,

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### **Figure 8.**

*Induction of C. elegans major stress or host defense responses by SiNP treatment. Fluorescent images of worms treated with H2O* (*a*), *SiNPs* (*b*)*, and tunicamycin* (*c*) *with a GFP reporter for ER stress. Fluorescent images of worms treated with H2O (d), SiNPs (e), and ethidium bromide (f) with a GFP reporter for mitochondrial stress. Fluorescent images of transgenic worms carrying the GFP reporter for oxidative stress subjected to H2O* (*g*)*, SiNPs (h), and H2O2 (i) treatment. Fluorescent images of transgenic worms carrying the GFP reporter for innate defense subjected to H2O (j), SiNP treatment (k), and physical injury (l). The same magnification was used in all of the images (m). Quantitative analysis of fluorescent intensity fold change of worms treated with H2O and SiNPs and a positive control group corresponding to (a–l). N ≥ 20. Error bars represent mean ± SEM; \*\* p < 0.01 [139].*

#### **Figure 9.**

*Possible mechanism of nanoparticle-induced mortality in adult Drosophila. (A) Location of spiracles in drosophila: sp1, mesothoracic spiracle; sp2, metathoracic spiracle; sp3 to sp9, abdominal spiracles, image from Lehnmann et al. (B) SEM image shows mesothoracic and metathoracic spiracle of an adult drosophila (blue square) Center row (C − E): SEM images of spiracles in unexposed drosophila; sp1 (C), sp2 (D), both 20–50 μm, and an abdominal spiracle (E) at 5 μm. Bottom row (F − H): Spiracles are covered/decorated with nanomaterials (see arrows) after dry exposure of adults to CB (F); MWNTs (G); CB (H) differential toxicity of carbon nanomaterials in drosophila: Larval dietary uptake is benign, but adult exposure causes locomotor impairment and mortality [140].*

vertebrate specific genetic disorder models cannot be developed in *D. melanogaster*. Other than this, unlike other models, amount of ingestion of NPs per flies cannot be accurately standardized as they are not gavage fed. Even after the above-mentioned limitations they remain one of the simplest models along with *C. elegans* to develop transgenic lines by breeding.

#### **4.2 Vertebrate models**

Vertebrate models for studying nanotoxicity include zebrafish, rabbit rodent models like mice, rat, hamsters etc. They share higher similarities with human beings in terms of the respiratory, circulatory, and nervous system. However, studying long term exposure of NPs in these organisms, especially rodents, becomes challenging due to their longer lifespan and gestation periods. The following section will briefly discuss the recent reports of using these vertebrate models for nanotoxicology studies and their limitations.

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### *4.2.1 D. rerio*

Zebrafish or *D. rerio* is one of the few vertebrate models with shorter breeding and offspring rearing period. This allows it to be a vertebrate model with ease to study developmental stages and the effect of NPs on life cycle. Also, oral and circulatory introduction of NPs is possible through zebrafish. The oral toxicity studies are conducted by introducing NPs in embryonic medium or fish water. The NPs are introduced in the blood stream directly using micro injections or intravitreal, intraperitoneal, intraventricular injections. Zebrafish models have been used to develop blood brain barrier models, tumor models and studied to see the effect and penetration properties of these nano formulations [146–150].

Toxicity of nanoparticle formulation is often assessed by survival analysis, cardiac rhythm studies, morphological changes in eye, spine and fin development, edema in cardiac sac etc. Behavioral changes like swimming pattern, response to tapping and light are also investigated. Like fruit flies, conservation of autophagy related genes to human beings allows using zebrafish models for studying the autophagy related gene expression with respect to nanoparticle treatment. Recently, treatment of ZnO nanoparticle in zebrafish model has displayed an increase in inflammation related gene over expression [151]. There are also reports of siler NPs affecting the gut microbiota of zebrafish [152]. Embryonic zebrafish studies have also reported the effect of silica based NPs in reducing the blood pressure in zebrafish and vascular endothelial cells (**Figure 10**) [153].

One of the major limitations of using zebrafish is the inability to use them for studying respiratory effects of aerosol based nano formulations. Other than this limitation they are also not ideal for breast cancer and prostrate cancer models as they lack the appropriate tissue of origin in their body architecture.

#### *4.2.2 Rabbit and rodent models*

Rabbits were classified as rodents till the early 20th century. They along with models with mice, rat and limitedly hamsters have been used for studying pharmacokinetics and pharmacodynamics of nanoparticle formulations. Their organs are also harvested and used for tissue distribution studies as they have striking similarity in tissue characteristics with human beings. Ocular and dermal toxicity of NPs are mostly studied in rabbits. Recently, silver NPs were tested on shaved skin regions on albino rabbits and the toxicity was analyzed using prefixed criteria. Dry skin, scaling in doses lower than 100 ppm and erythema in higher doses up to 4000 ppm was observed as part of this study [154]. Nano-hydroxyapatite was intravenously introduced to New Zealand white rabbits and it was noted that they does not affect liver function, and renal function in the animals [155]. In another study conducted to understand the toxicity of aflatoxin B1, treatment with curcumin and ZnO NPs prevented lipid and protein degradation via oxidation and showed better liver health as compared to aflatoxin B1 treated groups [156]. In corneal fibrosis (haze) model in rabbit using excimer laser performing -9D photorefractive keratectomy (PRK), nanoparticle formulation containing BMP7 topical application 5 minutes after PRK reduced the corneal haze by 50 percent with no toxicity [157]. Nephroprotective effects of NPs have also been studied using rabbit models. Studies using CaO NPs however have reported significant toxicity in liver and kidney after exposure [158].

Rodent models of nanotoxicity mainly includes rats and mice. Immune compromised, genetically altered rodents are very commonly used in nanotoxicology studies

#### **Figure 10.**

*Inflammatory response and vascular endothelial cell dysfunction induced by SiNPs. (a,B) SiNPs increased the recruitment and chemotaxis of neutrophils in caudal vein of Tg(mpo:GFP) zebrafish. (C,D) SiNPs inhibited the expression of vascular endothelial cells in Tg(fli-1:EGFP) zebrafish. n = 30, data are expressed as mean ± standard deviation from three independent experiments (\*p < 0.05). Scale bar: 100μm [153].*

and have been reviewed better in various book chapters and reviews elaborately [159–162]. Briefly they are used for central nervous system disorder models, cancer models, hepatotoxicity models, aerosol treatment models. They provide relatively more homology with human beings; however, the laboratory maintenance of rodents and rabbits are often challenging. Also, historical trait analysis with respect to nanoparticle formulation is often limited by the number of offspring produced and longer gestational periods. Besides this, there is no accepted duration of days or guidelines recommending the number of days nanoparticle exposure in rodents to be monitored. This largely leads to inaccuracies and inconstancies in reporting toxicity of the same or similar nano formulation by different research groups.

### *4.2.3 Primate models*

Primate models for nanotoxicity analysis rose to prominence after a study conducted on *Rhesus macaque* by intravenously injecting 25 mg/kg of phospholipidmicelle-encapsulated CdSe-Cds-ZnS QDs [163]. The study term was 90 days and the authors observed significant changes in behavioral patterns including loss of sleep, appetite, body weight, physical activity etc. By the end of study term, most of these effects were reversed to original state, however the particle displayed accumulation on liver and kidneys. Further studies on this were not carried out to understand the long term implications of hepatic accumulation. Quantum dots are generally considered to be safe to administer after conducting studies on *in vitro* and other *in vivo* models. However, this study indicated the need of behaviorally closer primate models to draw significant conclusion on nanoparticle toxicity before human trials.

Following this study, there were multiple reports of nanoparticle toxicity analysis using primates. Polylyisne conjugated DNA NPs for targeting retinal pigment

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

epithelium was studied using baboons showing no inflammatory response in the eye [164]. Also in cynomolgus monkeys, the safety of gadolinium based NPs for imaging purpose was evaluated [165]. In another studies, PEG-bl-PPS polymerosomes were found to be nontoxic in non-human primates [166]. Also, cargo of siRNA in cyclodextrin with transferrin ligands as targeting moieties were also found to be relatively safe in cynomolgus monkeys [167]. However, there were elevated levels of creatinine and nitrogen along with certain inflammatory cytokines at higher dosage. In another study, mice and *Rhesus macaque* were compared on its effect of CdSe/CdS/ZnS semiconductor NPs in placental crossing and miscarriage rates. Interestingly, in the rodent models there was not toxicity recorded even though the particles were shown to cross the placenta. There was no miscarriage in rodents and fetus displayed no abnormalities. However, in primate models there was a 60 percentage rate of miscarriage establishing the fact that primate models are far superior in compared to rodent models for toxicity analysis [168].

Even though they are ideally the closest to understanding human body's response to nano formulations, testing these formulations on them require more human resources and expertise along with stricter ethical guidelines in handling them. Also, these magnificent creatures are often sacrificed at the end of the study to harvest organs and understand tissue damage. Most of them being social organisms, this would have larger implications on their family group and could even lead closer ones to depression. Infant carrying is one such response displayed by mothers losing infants in primates. Secondary responses of curiosity and stress to death of infants or members is often displayed by these primates [169–172]. A closer evaluation of the morality and scientific rationale should be evaluated before conducting such studies.

### **5. Conclusions**

The past few decades witnessed the advent of nanoparticles and their potential use in multiple fields of biomedical sciences. From drug delivery to semiconductor devices, nanoparticles find applications around us. Informed use of nanomaterials, especially on its toxicity is highly relevant as more and more studies report the hazardous effects of these particles. The current chapter discussed *in vitro*, *in vivo*, *ex vivo* models for evaluating nanoparticle toxicity. As we analyze the plethora of assays conducted to study, in some cases, same particles in multiple model systems, we understand the varying toxicity reports. Such studies challenge the dangerous assumption of deeming a NP to be nontoxic by simply analyzing *in vitro* and in some cases rodent models. The need of non-human primate models closer to the genetic and physiological profile of human beings vs. the morale of sacrificing animal life for our benefit need to be carefully questioned. Alternate strategies like organ-on -chip models require further refinement and balance in incorporating parameters to better mimic study conditions.

### **Acknowledgements**

Authors acknowledge the financial support provided by Blazer foundation and Medical Biotechnology Program at Department of Biomedical Sciences, UIC College of Medicine Rockford.
