Nanomaterial-induced toxicity in pathophysiological models representative of individuals with pre-existing medical conditions

ABSTRACT

The integration of nanomaterials (NMs) into an ever-expanding number of daily used products has proven to be highly desirable in numerous industries and applications. Unfortunately, the same “nano” specific physicochemical properties, which make these materials attractive, may also contribute to hazards for individuals exposed to these materials. In 2021, it was estimated that 7 out of 10 deaths globally were accredited to chronic diseases, such as chronic liver disease, asthma, and cardiovascular-related illnesses. Crucially, it is also understood that a significant proportion of global populace numbering in the billions are currently living with a range of chronic undiagnosed health conditions. Due to the significant number of individuals affected, it is important that people suffering from chronic disease also be considered and incorporated in NM hazard assessment strategies. This review examined and analyzed the literature that focused on NM-induced adverse health effects in models which are representative of individuals exhibiting pre-existing medical conditions with focus on the pulmonary, cardiovascular, hepatic, gastrointestinal, and central nervous systems. The overall objective of this review was to outline available data, highlighting the important role of pre-existing disease in NM-induced toxicity with the aim of establishing a weight of evidence approach to inform the public on the potential hazards posed by NMs in both healthy and compromised persons in general population.

KEYWORDS:

• Engineered nanomaterials
• pre-existing disease
• toxicity
• inflammation
• oxidative stress
• genotoxicity
• hazard assessment

Introduction

The rapid expansion and exploitation of engineered nanomaterials (NMs) (a material with at least one dimension at the scale of less than 100 nm) has contributed to substantial interest in the fields of nanotechnology and nanomedicine (Brown et al. 2019; Kermanizadeh et al. 2021; Kermanizadeh, Powell, and Stone 2020; Vance et al. 2015; Verdon et al. 2022; Zhao and Castranova 2011). The worldwide nanotechnology market was valued at $2.4 billion in 2021 with over 2000 products incorporated with NMs in some form (Emergen Research 2022). However, the same unique physicochemical characteristics such as small size, charge, shape, and solubility, which make NMs desirable to many sectors might also exert an impact on their potential adverse effects. With the inevitable and continual rise of public and occupational exposure from increasing production and utilization of NMs, there is an imperative need to consider the possibility of potential detrimental health consequences of material exposure (Alaraby et al. 2016; Kermanizadeh, Powell, and Stone 2020; Laux et al. 2018). The hazard of NMs to human health manifests in a number of pathological outcomes including, but not limited to inflammation, oxidative stress, apoptosis, necrosis, autophagy, fibrosis, genotoxicity, and carcinogenesis (Demir 2020; Verdon et al. 2022).

With the increasing manufacture and use of NMs, human exposure to materials is inevitable. The skin, airways, and the gastrointestinal tract (GIT) are continually in contact with the external surroundings, therefore all three systems are primary exposure sites for NMs. However, it is now understood that NMs are able to translocate to a number of distal organs from the original point of entry (site of exposure) (Kermanizadeh et al. 2015b; Oberdorster et al. 2002. Pusiney, Baeza-Squiban, and Boland 2018). Dependant on route of exposure, varying quantities of NMs are released into the bloodstream and transported to secondary organs including but not limited to the cardiovascular system, liver, and the brain (Bai et al. 2014; Hullmann et al. 2017; Kermanizadeh, Powell, and Stone 2020; Sadauskas et al. 2009).

Any toxic/pathogenic effects of inhaled NMs are dependent on a significant lung burden, which is governed by rates of deposition and clearance. It is understood that clearance in the organ is influenced by physicochemical properties of the material (Muhlfeld, Gehr, and Rothen-Rutishauser 2008; Pusiney, Baeza-Squiban, and Boland 2018). The upper airways are predominately cleared by mucociliary transport, while alveolar clearance is conducted by pulmonary macrophages (Jo et al. 2020). The phagocytic clearance of NMs in the lungs is associated with release of pro-inflammatory mediators, generation of reactive oxygen species (ROS), and a host of other mediators (Feitosa et al. 2022; Murphy et al. 2016). This mode of toxic impact is predominantly associated with cases in which the deposition rate of NMs exceeds the clearance rate (Kim et al. 2021). It is believed that bio-persistent NMs might reside in the lung for longer periods of time and are associated with induction of adverse effects in this organ (Barua and Mitragotri 2014; Kim et al. 2021). Of note, oxidative stress is of particular importance in NM-induced toxicity (Capek and Rousar 2021; Demir 2022). It is understood that a range of NMs (dependent upon physicochemical properties) might generate intracellular ROS within a cell. In particular, the uptake of certain materials as well as dissolution and release of ions within a cell are believed to be extremely important factors in generating increased intracellular ROS levels. Mitochondrial dysfunction as evidenced by alterations in mitochondrial morphology, mitochondrial membrane potential and enhanced cytochrome C release were noted (Wu et al. 2020). Subsequently, NMs-induced elevated ROS might also result in NF-κB activation mediated inflammation and potentially cell death (Pang et al. 2021). Further, redox imbalance might result in genotoxicity within a cell. This increased ROS generation and recruitment of immune cells can in turn result in secondary oxidative stress and generation of free radicals in tissue resulting in further damage (Bonner et al. 2010; Kermanizadeh et al. 2015a; Kim et al. 2009; Liu et al. 2017; Møller et al. 2010).

The ingestion of NMs might occur directly from food, water, or orally administered medicines as well the mucociliary clearance of the airways. The bioavailability of NMs in the GIT is very complex due to (1) distinct pH in varying biological environment with the digestive tract (i.e. stomach as compared to the intestines), (2) presence of a mucus layer which trap non-soluble materials and (3) an abundance of digestive enzymes. This issue is further complicated by the fact that the physiochemical characteristics of different NMs influence their interactions with biological surroundings (Ma et al. 2014). In addition, the inherent characteristics of NMs may potentially influence how these substances are affected by pH, mucous, and GIT enzymes. Evidence indicates that significant quantities of ingested NMs are rapidly passed through the GIT and lost via feces (Kermanizadeh et al. 2015b). However, it is also believed that NMs in GIT enter the main circulation via microfold cells that cover the Peyer’s patches (McCright et al. 2021; Møller et al. 2012). There is uncertainty and contradictory data on the quantities of NMs that are released into the bloodstream following oral route of exposure (De Jong et al. 2019; Kermanizadeh et al. 2022a).

There is also some concern regarding NM-induced toxicity following dermal exposure (Adib et al. 2016; Ahmad 2021; Cao et al. 2021, 2016; Christophers and Schroder 2022; Ezealisiji and Okorie 2018; Gan et al. 2020; Larese et al. 2009; Monteiro-Riviere et al. 2011; Nohynek, Dufour, and Roberts 2008; Oberdorster et al. 2005; Raju et al. 2018; Teow et al. 2011). In this review, this route of exposure will not receive any further attention as it was not identified in any studies in which dermal exposure was utilized to investigate NM-induced toxicity in pathophysiological models.

In addition to the translocated NMs from pulmonary and GIT exposure, direct injection of materials loaded with drugs and diagnostic or imaging agents (nanomedicines) into the bloodstream is another principal route of NM entry into the circulatory system. As indicated previously, NMs in the blood distribute to a wide range of target organs as evidenced by almost 100% of non-surface modified particulates delivered via the intravenous route (IV) reaching the liver in a matter of min (Kermanizadeh, Powell, and Stone 2020). In systemic circulation, NMs may interact with plasma proteins, platelets, coagulation factors, erythrocytes, and leukocytes, which influence their biological behavior and eventual destination. The accumulation of NMs in various organs varies based upon their physicochemical properties, material coating, formation of protein corona in the biological milieu, route of exposure, bio-persistence of the material and of course the organ that is being investigated (Cortez-Jugo et al. 2021). As an example, unmodified non-soluble NMs introduced into the blood are readily opsonized and taken up by reticuloendothelial system and accumulate in the liver and spleen. This topic has been discussed in detail elsewhere (Bonner 2010; Kermanizadeh et al. 2015b).

In 2021, chronic diseases were estimated to be responsible for mortality in approximately 60 million people globally making up 7 out of 10 deaths. Importantly, a substantial proportion of the global populace numbering in the billions are currently suffering from a range of long-term health conditions including diabetes, chronic liver disease, asthma, chronic obstructive pulmonary disease (COPD) as well as cardiovascular complications. Further, it is not inconceivable that large numbers globally suffer from a wide spectrum of undiagnosed sub-clinical conditions. Due to the massive number of people affected, it seems logical that individuals with chronic disease are also considered and incorporated in NM hazard and risk assessment strategies.

In this review, all studies that involve NM-induced adverse health effects in in vitro and/or in vivo models which are representative of individuals in the general population with pre-existing medical conditions were examined. The focus of the review was on the pulmonary, cardiovascular, hepatic, gastrointestinal and central nervous systems. Thus, the aim was to determine NM-induced health effects in in vitro and/or in vivo models which are representative of individuals in the general population with pre-existing medical conditions by summarizing the relevant literature carried out between 2002 and 2022. The search criteria conducted in Web of Science included a combination of the following terms: “nanoparticles,” “nanomaterials,” “pre-existing disease,” “diseased models,” “pulmonary system,” “lungs,” “COPD,” “asthma,” “cardiovascular system,” “heart,” “liver,” “steatosis,” “non-alcoholic fatty liver disease,” “alcoholic liver disease,” “fibrosis,” “cirrhosis,” “central nervous system,” “brain,” “Parkinson’s disease,” “Alzheimer’s disease,” “gastrointestinal tract,” “Crohn’s disease,” “inflammatory bowel disease,” “diabetes,” “toxicity,” “cytotoxicity,” “adverse effects,” “inflammation,” “genotoxicity,” “DNA damage,” “oxidative stress,” “in vivo,” “in vitro,” “oral exposure,” “intravenous exposure,” “dermal exposure,” “intratracheal instillation” and “inhalation.” The last literature search was conducted on 21-5-2022. Due to the inevitable limitations and exclusionary nature of any literature search, studies that did not include the search terms in the title or did not provide adequate information in the abstract might have been unintentionally omitted. However, all relevant negative data from the studies in which NM-induced toxicity was investigated in disease models but not observed are included. All attempts have been made to make each study section comprehensive – it is hoped that this will allow for the overall message to be delivered coherently and conclusively. Any studies that did not provide adequate NM characterization data are excluded. This review only focused on non-modified particulates and engineered NMs and not nanomedicines or naturally occurring materials. Further, only manuscripts in English are included. From a toxicological standpoint, investigations that used high(er) non-physiological concentrations/doses were included as these still constitute a noticeable feature in the current nanotoxicology research landscape. Importantly, this review did not describe the disease initiation protocols in detail. Finally, the review did not cover cancers as pre-existing disease – the rationale for this decision is covered in the discussion section. The main body of the review is structured by different physiological systems and further segregated into in vivo and in vitro sections.

NM-induced toxicity in pathophysiological pulmonary models

Han et al. (2011) studied the toxicity of SiO₂ NMs in allergic asthma rodent models. In this investigation, ovalbumin (OVA)-treated (to induce pulmonary inflammation) and saline control 8-week-old Wistar rats were exposed to approximately 20 nm NMs for 30 days at daily doses of 4 or 8 µg via intratracheal instillation. Data demonstrated that the high dose NM exposure resulted in airway remodeling and aggravated inflammation which was significantly worse in the OVA-treated animals. Further, the IL4 levels in lung homogenates of NM-exposed animals was significantly higher in OVA exposed compared to controls. Han et al. (2011) concluded that the SiO₂ NMs were involved in the development and exacerbation of non-eosinophilic inflammation due to the Th1/Th2 cytokine imbalance as evidenced by increased labels of IL4.

The inflammatory response elicited by multi-walled carbon nanotube (MWCNT) exposure was investigated in control and common house dust mite (Dermatophagoides pteronyssinus) allergen primed 10–16-week-old C57BL6 mice. In this study, Carvalho et al. (2018) utilized a high nickel (Ni) content MWCNT (diameter of approximately 30 nm and a length between 5 and 15 µm). Following disease induction, mice were exposed to a MWCNT concentration of 2 mg/kg via intratracheal instillation. Data showed that animals sensitized with the allergen and further exposed to the MWCNT exhibited a significant rise in pulmonary inflammation compared to mice exposed to the allergen or NMs. Further, bronchoalveolar lavage fluid (BALF) differential cell counts demonstrated a marked influx of eosinophils, most notably in mice exposed to both allergen and NMs. This enhanced eosinophilic inflammatory response was also visualized via histology. Finally, in the diseased animals, there was significantly higher NM-induced cysteinyl leukotrienes (family of inflammatory lipid mediators synthesized from arachidonic acid) biosynthesis as compared to negative controls, allergen alone or MWCNT alone exposed animals. Taken together, results demonstrated that MWCNT toxicity was markedly exacerbated in the allergic models (Carvalho et al. 2018).

In another study, the effects of lipopolysaccharide (LPS) pre-exposure on the pulmonary toxicological effects of MCWNT (10–30 nm width and 0.3–50 mm length) was investigated. Cesta et al. (2010) used 7-9-week-old Sprague-Dawley male rats exposed to LPS via nasal aspiration which was followed by intratracheal instillation of NMs at a dose of 4 mg/kg. The experiment also included control groups which were administered PBS rather than LPS. Cesta et al. (2010) observed pulmonary fibrosis 21 days following MWCNT exposure. LPS pre-treatment alone produced no marked fibrosis but enhanced MWCNT-induced toxicological effects as evidenced by increased cell death frequency and elevated platelet-derived growth factor (PDGF)-AA (mediator of fibrosis) levels in lungs of diseased animals. Following the in vivo observations, Cesta et al. (2010) further showed that LPS enhanced MWCNT-Induced PDGF-A mRNA expression in lung macrophages (NR8383 cell line) and increased PDGF-Ra levels in primary rat lung fibroblasts in vitro. Overall data indicated that LPS stimulated inflammatory state exacerbated MWCNT-induced lung fibrosis by elevating production of PDGF-AA in macrophages and epithelial cells as well as increasing PDGF-Ra levels on pulmonary fibroblasts.

In a 2010 investigation, the acute pulmonary toxicological response was examined following a single dose (40 mg/kg) intraperitoneal administration of approximately 20 nm TiO₂ NMs in healthy and LPS primed male BALB/c mice (Moon et al. 2010). Following a 4-hr exposure, Moon et al. (2010) found a significantly increased influx of neutrophils and higher levels of TNF-α, IL1-ß, and MIP-2 in BALF of exposed animals. In addition, NM exposure resulted in activation of c-Src and p38 MAP kinase, in alveolar macrophages and NF-κß pathway in pulmonary tissue. Importantly, almost all NM-induced inflammatory parameters described above were significantly elevated in LPS primed mice. The only exception to this was TNF-α levels being lower in NM exposed animals in the disease model as compared to the non-primed rodents (Moon et al. 2010).

In an important inhalation study, Kim et al. (2017) subjected 9-week-old female OVA sensitized mice (species not specified) to 50 µg/m³ of aerosolized TiO₂ NMs in an inhalation chamber for 2 hr per day for 3 consecutive days. Data demonstrated that hyperresponsiveness in the airways and inflammation was increased in OVA sensitized animals, and these toxicological end-points were exacerbated by exposure to TiO₂ NMs as evidenced by elevated neutrophil, eosinophil, and lymphocyte counts in BALF. In addition, in the asthma model, the animals exhibited significantly higher IL-1β, IL18, NLRP3, and caspase-1 expression levels compared with saline-treated mice which was exacerbated by TiO₂ NM treatment. Overall data indicated that inflammasome activation might be at least partially responsible for toxicity in the mouse model of allergic asthma following TiO₂ NM exposure (Kim et al. 2017).

Exposure of TiO₂ (approximately 170 nm) NMs to OVA sensitized 12-week-old-BALB/c female mice was undertaken by Mishra et al. (2016). In these experiments, OVA sensitized or control animals were exposed to the NMs at four consecutive daily doses of 100 µg/per animal by intraperitoneal injection. The pulmonary toxicity data as measured in terms of total protein and lactate dehydrogenase (LDH) activity noted major lung injury both in OVA-treated and OVA plus NM treated which were approximately 1.6-fold and approximately 4-fold higher, respectively, compared to saline controls. Further, NM exposure increased Th₂/Th₁ immune response in asthma pathophysiology as demonstrated via significantly higher levels of IL4, IL5, and IL13 (Th2) and IL6 and TNF-α (Th1). These findings were further strengthened by upregulation of levels of Stat3, NF-kB in NM exposed asthmatic mice. Mishra et al. (2016) concluded that the TiO₂ NMs exacerbated the inflammatory response in lungs of pre-sensitized allergic animals and that these alterations are potentially regulated via the NF-kB pathway.

In another investigation, 8-week-old female BALB/c mice were sensitized intraperitoneally with OVA prior to exposure to two differently sized TiO₂ materials (fine TiO₂ – < 5 µm and nano TiO₂ – ~ 50 nm) three times a week for 2 hr for a 4-week period in an inhalation chamber at an exposure concentration of 10 ± 2 mg/m³ (Rossi et al. 2010). These experiments also included non-allergic control animals. In the asthmatic mice, there was increased numbers of eosinophils and lymphocytes in the airways compared to healthy controls. This was also accompanied by enhanced mucus secretion. Interestingly, and in contrast to the main body of literature, the numbers of eosinophils and lymphocytes both characteristic features of allergic inflammation were reduced in asthmatic mice after exposure to TiO₂ materials. The authors Rossi et al. (2010) further noted an inhibition of proinflammatory (IL1-ß, TNF-α) and Th2 cytokines (IL4 and IL13) in the lungs of asthmatic mice exposed to the materials.

In another study investigating the influence of SiO₂ NMs (approximately 10–20 nm primary size – agglomerates not fully characterized) on function impairment and exacerbation of pre-existing lung disease, OVA treated and saline control 12-week-old male Wistar rats were administered daily via intratracheal instillation 4 and/or 8 µg of material for 30 days. Han et al. (2011) reported airway remodeling in NM exposed animals with these effects being augmented in the diseased animals. Further, SiO₂ NMs at the highest dose induced a marked IL4 response which was greater than in OVA alone sensitized rats (Han et al. 2011).

Similarly, 11-week-old OVA sensitized female BALB/c mice were exposed to differently sized amorphous SiO₂ materials (diameter of 30, 70, 300 or 1000 nm) intranasally at doses of up to 250 µg/animal. The animals were exposed to a single dose of the materials and sacrificed at day 21 post exposure. Data demonstrated that the smallest diameter NMs at the highest dose induced the greatest significant IgE and IgG1 response in the diseased animals, which subsequently was markedly higher than controls (Yoshida et al. 2011). Further, Yoshida et al. (2011) found that the splenocytes from mice exposed to OVA and the 30 nm SiO₂ NMs secreted the highest levels of Th2 cytokine IL4 and IL5. The levels of these proteins were considerably higher than the asthmatic mice not exposed to the NMs and healthy controls.

Brandenberger et al. (2013) utilized 8-week-old female BALB/c mice which were co-exposed to OVA and an increasing up to 400 µg/animal intranasal dose of SiO₂ NMs (approximately 90 nm). In total, the animals received the NMs on four occasions over 8 days (day 1, day 3, day 6 and day 8). The animals were sacrificed on day 24. Brandenberger et al. (2013) noted that exposure to NMs during OVA sensitization resulted in an exacerbation of allergic airway disease as compared to OVA in healthy or NM-only exposed animals. The adjuvant effect observed was enhanced with increased NM dose. As an example, the total BALF cell numbers were 2.5-fold higher in NMs/OVA-treated mice compared to OVA alone. In addition, the adjuvant effect was most notable for the number of eosinophils being 25-fold higher in the co-exposed mice. The augmented toxicity also included higher serum IgE levels, mucous cell metaplasia, and Th2 (IL4, IL5, IL13) and Th17 (IL6, IL17a) cytokine protein secretion (Brandenberger et al. 2013).

In a similar study to Brandenberger et al. (2013), Han et al. (2016) examined the toxic and adjuvant effects of SiO₂ NMs in allergic airway inflammation mouse model. In this experiment, 6-week-old female BALB/c mice were co-exposed intranasally with OVA and three types of SiO₂ NMs (spherical – ~ 100 nm, mesoporous – ~ 100 nm and polyethylene glycol-conjugated materials – ~ 400 nm) on six occasions (10 mg/kg per treatment) over a period of 14 days. Han et al. (2016) demonstrated that the spherical/OVA and mesoporous/OVA exposure resulted in significant airway hyper-responsiveness as compared to NM only and saline/OVA exposed animals. The BALF cell counts also exhibited the same pattern with number of total cells, macrophages, lymphocytes, eosinophils, and neutrophils significantly increased in the spherical/OVA and mesoporous/OVA exposed mice. Further, histological analysis showed peribronchial and perivascular inflammation and goblet cell metaplasia in spherical/OVA-administered groups but not visible in other treatment animals. Finally, in the spherical/OVA-treated animals, levels of IL5, IL13, IL-1β, and IFN-γ, were significantly elevated compared to controls and markedly higher than in other treatments including NMs only and OVA only (Han et al. 2016).

In another investigation, Chuang et al. (2013) examined the toxicity of inhaled Ag NMs (approximately 30 nm) in allergen-sensitized mice. In this study, OVA sensitized, and control female BALB/c mice (8-weeks-old) were exposed to 3.3 mg/m³ NMs in an inhalation chamber for 7 consecutive days (6 hr per day). The animals were euthanized 5 days following last material exposure and BALF, blood and tissue samples were collected. Data showed that Ag NM exposure induced a significant elevation in neutrophil, lymphocyte, and eosinophil infiltration in the lungs which was accompanied by higher levels of IgE, leukotriene E4, IL13 and oxidative stress marker (8-hydroxy-2′-deoxyguanosine [8-OHdG]) in both healthy and allergic mice. Importantly, there was no significant difference in the toxicity as depicted by biomarkers listed above observed following NM exposure between healthy and asthmatic mice. The findings from this study seem to suggest that Ag NMs were of concern both in healthy and asthmatic mice models (Chuang et al. 2013).

The inflammatory properties of a PEGylated and citrated Au NMs (core size 5 nm) were investigated in healthy and asthmatic mice models. Female (6-8-week-old) wild-type BALB/c mice were sensitized with OVA for three days prior to intranasal exposure to NMs via two administrations on day 1 and 7 (the doses were not stated). Omlor et al. (2017) demonstrated that both Au NMs induced an anti-inflammatory effect in asthmatic mice as evidenced by reduction of total cell, macrophage, and eosinophil counts compared to healthy animals. Omlor et al. 2017) postulated that a reason for this might be due to the physical binding of Au NMs to pro-inflammatory cytokines resulting in their subsequent inactivation. Further, data showed higher level of lymphocytes and neutrophil counts in BALF of NM-exposed asthmatic animals compared to NM-administered healthy animals and asthmatic non-exposed animals. Finally, and importantly, Omlor et al. (2017) noted that pre-existing asthma increased NM uptake in pulmonary cells as well as systemic translocation most predominant in heart and the liver; with accumulation considerably higher in the diseased compared to the healthy mice.

In another study, the toxicity of two differently sized Fe₂O₃ NMs (approximately 35 nm and 150 nm) was assessed in the OVA sensitized and healthy 7-week-old BALB/c mice. In this experiment Ban et al. (2013) intratracheally administered the materials were on 4 occasions over 12 hr at doses of 100, 250, or 500 µg/animal before and during OVA sensitization. The animals were sacrificed 21 days after the final exposure. Results from the study showed that exposure to the larger NM at the higher doses of the NMs significantly inhibited the Th2 dominated allergic response in the asthmatic mice as evidenced by lower eosinophil numbers in the BALF of exposed animals which was accompanied by decreased IgE levels. However, exposure to low dose of 35 nm Fe₂O₃ NMs in the diseased animals had an adjuvant effect on the Th2 response. The suppression of the allergic Th2 responses by the Fe₂O₃ NMs within this study may be due to the possibility of rise in number of plasmacytoid dendritic cells (immune cell that are known to secrete large quantities of type 1 interferons) which has attributed anti-Th2 response properties. On the other hand, the adjuvant effects of low-dose exposure of the smaller Fe₂O₃ NMs are potentially attributed to enhanced particle phagocytized macrophages resulting in enhanced antigen presenting function of dendritic cells by alternatively activated macrophages subsequently leading to pronounced T-cell activation and Th2 response (Ban et al. 2013).

In a 2015 study, the focus was placed on understanding the toxicity of approximately 50 nm ZnO NMs (low-dose agglomerates approximately 180 nm and high-dose agglomerates and approximately 350 nm) in healthy and asthmatic mice. Huang et al. (2015) employed 7-week-old female BALB/c mice which were administered NMs on two occasions (day 1 and day 7) at doses of 1 mg/kg and 5 mg/kg via oropharyngeal aspiration. Following the last NM exposure, animals were sacrificed at days 1, 7 and 14. Huang et al. (2015) reported that the BALF total cell counts and eosinophil numbers were more prominent after exposure to the combination of ZnO NMs in the asthmatic animals as compared to the rest of the exposure groups (PBS, asthma, or ZnO NM exposure in healthy mice). Histological examination of the lung sections displayed that treatment of ZnO in healthy and asthmatic mice resulted in eosinophil infiltration in peribronchial, peribronchiolar, and perivascular interstitial tissues and alveolar space. However, there was no marked difference in the pathology observed between healthy and asthmatic animals exposed to ZnO NMs (Huang et al. 2015).

In one of the earliest sensitization studies concerning NMs, 6-week-old male ICR mice were exposed to OVA every two weeks for 6 weeks (Koike et al. 2008). The experiment included PBS controls for the same period of time. The NM group received 50 µg of approximately 15 nm or 55 nm carbon black NMs every week for 6 weeks via intratracheal instillation. The sensitized groups received the combined treatment of OVA and NMs. The numbers of total cells in the lung were significantly greater in combined 15 nm NMs and OVA and 15 nm NMs only mice compared to vehicle control. Similar patterns were observed, in terms of increased number of dendritic cells, macrophages, and B cells in BALF. However, there was no significant alteration with respect to pre-existing disease in these cell counts (Koike et al. 2008).

In a study, Kroker et al. (2015) utilized 8-week-old female BALB/c mice were treated with 2.5 mg/kg 15 nm carbon black NMs (approximately 850 nm agglomerates) with or without ectoine (osmolyte with membrane stabilizing and inflammation reducing agent) via pharyngeal aspiration for a total of 4 doses over the first 9 days of the experiment. Animals were sensitized by repeated administration of OVA over the whole experimental period of 35 days and BALF, blood, and lymph nodes were collected. Kroker et al. (2015) found significantly higher levels of IgE in the NM sensitized animals as compared to healthy animals which was further prevented by ectoine. Further, data showed enhanced neutrophilic lung inflammation induced by carbon NMs during allergic sensitization (Kroker et al. 2015).

In an investigation by Ryman-Rasmussen et al. (2009) 6-8-week-old male C57BL6 mice were OVA sensitized prior to exposure to MWCNTs (0.5–40 µm) in a nose-only inhalation chamber at a dose of (approximately 100 mg/m³) for a 6-hr period before sacrifice 1 or 14 days after NM exposure. At 14 days post NM exposure, the histopathology of lung tissue exhibited an elevation in collagen deposition in the reticulum around the basement membrane of the bronchioles in the sensitized mice relative to unsensitized animals that inhaled the same dose of MWCNT as well as the sensitized and unsensitized saline controls. The OVA challenge increased IL13 and TGF-ß1 protein levels in the BALF. However, these levels were not significantly different following NM exposure. Finally, IL5 mRNA levels in homogenized whole-lung tissue were significantly higher in diseased mice exposed to the NMs, but not in OVA or MWCNT alone exposed animals. Data suggest that the risk of MWCNT-induced fibrosis is greater in pre-existing allergic inflammation indicating that individuals with asthma might be more susceptible to MWCNT toxicity that non-asthma sufferers in general populations (Ryman-Rasmussen et al. 2009).

In another study, healthy and OVA sensitized 7-week-old male ICR mice were exposed to MWCNT (approximately 65 nm; surface area, 26 m²/g; carbon purity, 99.8%; fiber length not specified) once a week for a total of 6 weeks via intratracheal instillation where each NM dose was 50 μg/animal (Inoue et al. 2009). Concurrently, OVA-sensitized T-cells isolated from the spleens of allergic mice were exposed to MWCNTs at concentration range of 0.1–1 μg/ml as well as 10 ng/ml recombinant mouse granulocyte macrophage-colony stimulating factor. The results showed aggravated inflammation in the lungs of diseased animals compared to control mice as evidenced by increased BALF total cell, neutrophil, and eosinophil numbers. Further, histological analysis demonstrated that in combined treatment with OVA and NMs increasing numbers of neutrophils and eosinophils was detected accompanied by lymphocyte sequestration into the lung parenchyma. Similarly, MWCNT exposure in asthmatic animals resulted in an elevated number of goblet cells in the bronchial epithelium which was not observed in the other treatment groups. The MWCNT and OVA exposure also amplified lung protein levels of inflammatory cytokines and chemokines, namely, IL4, IL5, IL13, IL18, IL33, and IFN-Ƴ. Finally, the in vitro exposure of MWCNT significantly enhanced allergen-specific syngeneic T-cell proliferation. Data from this study suggest that MWCNTs might potentially intensify allergic airway inflammation (Inoue et al. 2009).

Another group Mizutani, Nabe, and Yoshino (2012) investigated the adverse effects of MWCNT (diameter approximately 15 nm and length 0.1–10 µm) exposure on ovalbumin sensitized 7-week-old BALB/c mice. The animals were sensitized on days 0, 1, 2, 14, 15, and 16 with saline, 0.01–1 mg MWCNT/ml (30 µl per dose), or OVA and NMs via intranasal route. Further, to achieve systemic sensitization mice were exposed by intraperitoneal injection with OVA on days 0 and 14. The sensitized mice were challenged on days 28, 29, 30, and 35 with 1% OVA by intratracheal administration. In the NM exposed asthmatic animals, early and response airway resistance was noted that was higher than all other treatment groups. Data demonstrated that OVA sensitization in conjunction with MWCNT exposure promoted airway inflammation as evidenced by increased total cell, macrophage, neutrophil, and eosinophil numbers and goblet cell hyperplasia in the lung compared with the vehicle, MWCNT or OVA treated mice. Further, the NM-administration in the diseased animals resulted in highest IgE, IgG1 and IG2a levels in serum and elevated levels of IL4, IL5, IL15, and IL17 in lung tissue homogenates (Mizutani, Nabe, and Yoshino 2012).

The toxicity of CeO₂ NMs (approximately 25 nm and agglomerates of approximately 100 nm) was assessed in animals in the presence and absence of the common allergen house dust mite and diesel exhaust particles. In these experiment Meldrum et al. (2020) used 6-8-week-old female BALB/c that received the diesel exhaust materials (1.25 mg/kg), CeO₂ (2.5 μg/kg or 75 μg/kg) and dust mites repeatedly via intranasal instillation over a period of 19 days (9 doses) with the animals sacrificed on day 20. The results showed total cell number was increased in the sensitized animals and further modified by the higher dose of CeO₂ NMs as noted by augmentation of pulmonary inflammatory response. Interestingly, the repeated administration of CeO₂ NMs in the sensitized animals significantly elevated the levels of airway mucin, eosinophils, lymphocytes, IL5, IL13, IL7A and plasma IgE which were significantly higher than in the diesel and house dust mite exposed animals alone (Meldrum et al. 2020).

Two years earlier Meldrum et al. (2018) examined the impact of CeO₂ NMs (approximately 25 nm and agglomerates of approximately 170 nm) exposure in 8-week-old female BALB/c model of asthma using house dust mite induction. Meldrum et al. (2018) utilized the same experimental protocol for intranasal administrations and material doses as described in Meldrum et al. (2020). Data demonstrated that co-exposure of CeO₂ NMs at the higher dose with house dust mite further increased total BALF cell counts above the dust mite alone induced animals. Importantly, CeO₂ NM exposure in the absence of sensitization did not result in a significant change in any BALF cell counts. Meldrum et al. (2018) also detected that mast cell numbers, eosinophils total plasma IgE and goblet cell metaplasia were significantly elevated in the high dose CeO₂ NM exposed sensitized animals compared to the rest of the treatment groups and controls. This was accompanied by increases in IL-4, CCL11 and MCPT1 gene expression. Collectively data suggest the onset of a type II inflammatory response. Overall, data clearly showed that NM exposure augments and modulates the pulmonary inflammatory response toward a type II milieu (Meldrum et al. 2018).

Ko et al. (2020) examined the potential toxic effects of SiO₂ NMs (primary size approximately 50 nm and aggregates of approximately 440 nm) in an OVA-sensitized asthmatic mice model. In these trials, 6-week-old- female BALB/c mice were divided into different treatment groups: vehicle control, OVA sensitization only, healthy mice exposed to materials and sensitized animals exposed to NMs. The material exposed animals received the SiO₂ via intranasal instillation at doses of 5, 10 or 20 mg/kg on three occasions every other day over a 5-day period. Mice exposed to SiO₂ NMs in the diseased animals displayed the highest and significant inflammatory cell counts mostly notable for eosinophil total cell counts, IL1ß, IL5, IL6, IL13 and TNF-α levels, mucus secretion and nucleotide-binding and oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) levels which were well above the other treatment groups. Evidence indicates that NMs adverse effects are aggravated in the asthma models compared to the healthy animals (Ko et al. 2020).

In another study focusing on SiO₂ NMs (spherical, mesoporous, and PEGylated – all approximately 100 nm), 6-week-old healthy or 8-week-old OVA sensitized BALB/c female mice were intranasally exposed to the materials on 3 consecutive days at a dose of 200 µg per animal for a total 600 µg in the three exposures. Park et al. (2015) reported that acute NM exposure induced significant airway inflammation as evidenced by total cell count, neutrophil and basophil counts in the BALF which was aggravated in the asthmatic model (most evident for the spherical NM). Further, significantly more cellular infiltration in peribronchial and perivascular tissues was observed in the NM-exposed diseased animals compared to the healthy NM-administered mice. In conclusion, the observations from this study demonstrated the adverse effects of SiO₂ NM exposure was significantly aggravated in asthmatic animals compared to healthy animals (Park et al. 2015).

In 2020, OVA sensitized 6-week-old female BALB/c mice were used to investigate the effects of TiO₂ NMs (approximately 50 nm). The experiments also included OVA only and a group containing healthy mice which were exposed to TiO₂ materials. The appropriate exposure groups received TiO₂ NMs at a dose of 200 μg/m³ in an inhalation chamber for 2 hr. Kim et al. (2020) found that differential inflammatory cell counts in the BALF in terms of total cells, macrophages, eosinophils, neutrophils, and lymphocytes were significantly aggravated byTiO₂ NM exposure in the asthmatic animals compared to healthy counterparts. Histological examination further showed that NM exposure in the diseased animals aggravated inflammatory cell infiltration and exudative alterations in the peribronchial layers and intraluminal areas of the bronchi compared to the healthy mice exposed to the same material at the same dose. Kim et al. (2020) concluded that TiO₂ NM-induced adverse effects are significantly augmented and aggravated in the asthmatic model as compared to healthy mice.

In a unique study, the effects of TiO₂ NM (approximately100 nm) exposure alone or respiratory syncytial virus and TiO₂ NMs on bronchial epithelial cells were investigated. In this study, Smallcombe et al. (2020) used immortalized human bronchial epithelial cells (16HBE) cultured on collagen-coated Transwell-permeable supports under liquid-liquid conditions. In addition, primary human bronchial epithelial cells isolated from the lungs of paediatric patients were cultured on Transwell membrane inserts under air-liquid interface conditions. Subsequent exposures were only performed when the polarized monolayers exhibited a trans-epithelial electrical resistance (TEER) reading >500Ωxcm². Following polarization of the monolayers, appropriate cells were infected with red fluorescent protein-labelled virus. The in vitro NM concentrations utilized were 10–100 µg/cm² and the exposure period was 48 hr. Furthermore, 6-8-week-old female C57BL6 mice were intranasally instilled with a single dose of TiO₂ NM (10–60 µg/mice) in the presence or absence of the virus. The animals were sacrificed on day 4 post-virus inoculation. Both in vivo and in vitro experiments contained all appropriate controls. Data showed that TiO₂ at the high doses induced pical junctional complex disassembly and increased paracellular permeability in both the immortalized and the primary human bronchial epithelial cells. However, these effects were significantly aggravated in the viral disease model compared to healthy cells. Further, exposure to TiO₂ NMs at 50 µg/cm² resulted in significantly elevated pro/anti-inflammatory cytokine/chemokine levels IL-1α, IL-1ß, IL2, IL4, IL5, IL10, IL12, IL13, TNF-α, IFN-Ƴ, CCL3, CCL4, CXCL10 and GM-CSF in the virus model as compared to PBS, virus alone or TiO₂ alone exposure groups. In vivo data demonstrated that leukocyte quantification and BALF total protein concentrations were significantly higher in the TiO₂ NM exposed disease model compared to healthy animals exposed to TiO₂ NMs or virus infected animals without NM exposure. Histological examination of the pulmonary tissue revealed a greater inflammatory response after co-exposure to virus and the NMs compared with virus infection or TiO₂ NM exposure alone. Finally, the same pattern was observed for significant elevations in levels of inflammatory cytokines/chemokines following co-exposure which included proteins IL1, IL6, TNF-α, IFN-Ƴ, CCL2 CCL5, leukemia inhibitory factor, and CXCL10 (Smallcombe et al. 2020).

In an interesting in vitro investigation, primary splenic leukocytes were exposed to complete medium, OVA or OVA plus carbon black NMs (approximately 20 or 40 nm) via a single dose of up to 12.5 µg/ml for 72 hr. Lefebvre et al. (2014) found changes in gene generation where co-incubation with the highest NM dose significantly increased IL4, IL10 and IL13 levels while there was a down-regulation of STAT-4 required for development of Th1 cells from naive CD4 cells. The overall NM-mediated inflammatory response was higher in sensitized cells compared to cells cultured in medium control. These alterations described above were consistently higher for the smaller of the two NMs (Lefebvre et al. 2014).

In a recent simple ex vivo investigation, the toxic properties of ZnO NMs (approximately 150 nm) were examined using primary lymphocytes isolated from healthy individuals or those suffering from asthma or COPD. The leukocytes were exposed to the NMs at concentrations of 10, 20 or 40 µg/ml for 6 or 12 hr. Kumar et al. (2015) observed no significant differences in cytotoxic effect of ZnO NMs on the lymphocytes of healthy and different lung disease patients

Finally, in the scrutinization of the literature two additional manuscripts (Enright et al. 2013; Jonasson et al. 2013) were identified which may have been included in this section but unfortunately, it was not possible to access the full articles and a response from the corresponding authors following requests for the manuscripts was not received.

NM induced toxicity in cardiovascular disease models

In an extremely interesting study, the toxic properties of superamagnetic iron oxide NMs were investigated in hyperlipidaemic models of atherosclerosis by Segers et al. (2022). In these trials, normolipidemic and hyperlipidaemic RAW264.7 (murine macrophage cell line) cells were exposed to dextran-coated small superparamagnetic iron oxide (approximately 120–180 nm) or ultra-small superparamagnetic iron oxide NMs (approximately 30 nm) at a dose of 100 µg Fe/ml for 24 hr. Concurrently, Segers et al. (2022) conducted 4 separate in vivo experiments in which a) Apolipoprotein E knockout (ApoE^−/−) mice received a single dose of dextran-coated small superparamagnetic iron oxide NMs (0.3 mg Fe/kg), or ultra-small superparamagnetic iron oxide NMs (1 mg Fe/kg) after 9 weeks on a western-type diet; b) 12-week-old low-density-lipoprotein (LDL) receptor knockout mice were fed on a western diet for 3 weeks prior to receiving dextran-coated small superparamagnetic iron oxide via the intravenous (iv) route at a dose of (1 mg Fe/kg); c) LDL receptor knockout mice were fed on a western diet for 14 weeks before administration of an antioxidant (EUK-134) intraperitoneally 1 hr prior to NM exposure as described above and d) LDL receptor knockout mice were fed on western diet for 9 weeks before receiving an iv dose of dextran-coated ultra-small superparamagnetic iron oxide NMs. For all the in vivo experiments, mice were euthanized 24 hr after the final NM injection. Data demonstrated that NM exposure increased apoptosis of foam cells compared with normolipidemic RAW macrophages in a time- and LDL concentration-dependent manner. The in vitro observations were confirmed in ApoE^−/− mice fed on a western diet, where apoptosis was not only limited to atherosclerotic plaques but also in the liver and spleen of the ApoE^−/− mice. Further, electron microscopy showed accumulation of superparamagnetic iron oxide nanoparticles in human carotid atherosclerotic lesions. Overall, these data illustrated that NM treatment enhanced apoptosis in atherosclerotic lesions and other macrophage-rich tissue in hyperlipidaemic mice. Further, antioxidant treatment prevented iron oxide NM-induced apoptosis both in vitro and in vivo (Segers et al. 2022).

The possibility of pulmonary inflammation accelerating NM-induced atherosclerotic plaque progression was also investigated. In this study, Christophersen et al. (2016) employed10-week-old female C57BL6 ApoE^−/− mice which were administered LPS by intratracheal instillation prior to similar instillation of two doses of carbon black (8.53 or 25.6 μg/mouse) once a week for 10 weeks. These experiments also included control groups in which mice did not receive LPS challenge. Results showed that progression of atherosclerosis in the aorta of ApoE^−/− mice after repeated exposure to NM and LPS was significantly higher than the NM only group (Christophersen et al. 2016).

In an investigation Ferdous et al. (2022) determined in 10-week-old male and female BALB/c mice (model representative of healthy individual) and angiotensin II exposed mice (a model for hypertension), the toxicity of approximately 40 nm PEGylated Au NMs. In these trials, the animals received a dose of 0.5 mg/kg of the NMs via intratracheal instillation on 4 occasions (on days 7, 14, 21, and 28) post-angiotensin II or vehicle infusion and were sacrificed on day 29. It was noted that NMs produced significant shortening of the thrombotic occlusion time in the arterioles and the venules of the diseased mice as compared to controls. In addition, the prothrombin time and activated partial thromboplastin time were exacerbated following NM exposure in hypertensive mice compared to healthy NM exposed animals. A similar pattern to the above was observed for the concentrations of fibrinogen, plasminogen activator inhibitor-1 Finally, the plasma levels of nitric oxide and superoxide dismutase (SOD) were markedly increased in the diseased animals as compared to healthy mice exposed to the materials at the same dosage regimen. The observations from this study demonstrate enhanced NM-induced adverse effects in the hypertension models (Ferdous et al. 2022).

The potential toxicity of ZnO NMs (approximately 100 nm) on healthy cardiovascular system or cardiovascular disease was investigated by Nagarajan et al. (2022). in a fertilized egg model. In this experiment, in ovo administration of the material at concentrations of 10, 20 or 40 μg/egg was undertaken. With the materials injected from day 8 to 12 on alternate days. The disease models (cardiac hypertrophy) were generated by co-exposure of ZnO NMs at the doses stated above with 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). After incubation for 12 days, the heart rate was measured and the heart from the chick embryos removed. The organ weight was recorded as a means of assessing cardiac hypertrophy. The findings showed that exposure to ZnO NMs at the concentration of 40 μg/egg significantly reduced development of blood vessels and elevated thiobarbituric acid reactive substances (TBARS) in heart tissue. Interestingly, however, exposure to NMs in the disease model resulted in significantly increased heart weight and hypertrophic responses compared to both the ZnO exposed healthy model and the AAPH alone exposure group (Nagarajan et al. 2022).

Metabolic syndrome is an established risk factor for development of chronic diseases including diabetes and cardiovascular disorders (Galassi, Reynolds, and He 2006). Kobos et al. (2020) induced metabolic syndrome in mice by exposure to a high 60% kcal high fat diet for 14 weeks. Subsequently silver nanoparticle at 20 nm diameter and at a concentration of 2 mg/kg iv via the tail vein was administered to metabolic syndrome mice and animals sacrificed after 24 hr. Silver nanoparticles decreased inflammatory gene expression CXCL1, Il-4, and Il-3 expression in spleen. In contrast gene expression in spleen of TNF-α, CXCL1, TGB-β, HO-1 and IL-4 in healthy mice. It is noteworthy that silver nanoparticles localized primarily in spleen and liver following iv injection. Data demonstrated that metabolic syndrome. which is a risk factor for cardiovascular diseases, influences the inflammatory responses induced by silver nanoparticles.

In an in vitro study, the toxic properties of a PEGylated Au nanorod (length approximately 50 nm, diameter approximately 25 nm) were investigated in human umbilical vein endothelial cells with or without LPS stimulus at concentrations of 10 µg/ml of NMs for 24 hr (Li et al. 2019). No significant influence of LPS in terms of NM-induced cytotoxicity or in material uptake was detected. However, Li et al. (2019) noted that KLF2 (Kruppel-like factor a transcription factor important for maintenance of function of vascular walls) was significantly down-regulated by treatment with LPS. Further, mRNA levels of MCP-1 were almost 8-fold higher in LPS and NM-treated cells as compared to NMs alone (Li et al. 2019).

Finally, one additional manuscript (Chen et al. 2008) was identified that focused on the effects of NMs in disease models of cardiovascular system but unfortunately, it was not possible to access the full article and a response from the corresponding authors following a request for the manuscripts was not received.

NM induced toxicity in pathophysiological gastrointestinal models

In a recent in vitro study, a triple cell Transwell culture system composed of differentiated Caco-2 (intestinal epithelial cells), HT29-MTX-E12 (goblet cells) and differentiated THP-1 cells (macrophages) as a mimic for inflamed intestine, were utilized to assess the adverse effects of polyvinylpyrrolidone-capped silver (agglomerates approximately 250 nm) and TiO₂ (agglomerates approximately 330 nm) NMs. Importantly, Kämpfer et al. (2021). used stable and inflamed (IFN-γ primed) variants of the triple cultures in these experiments. Following the 21 days to establish the system, cells were exposed to NMs on 5 subsequent days at doses of up to 30 µg/cm² for each treatment. Data showed sufficiently higher LDH activity release as means of measuring cell membrane integrity and inflammatory cytokine secretion IL6, IL8 and TNF-α following exposure to both materials which was not dose dependent. Kämpfer et al. (2021). did not observe a significant difference in the NM-induced toxicity between healthy and inflamed models in this study.

In another study by the same group Busch et al. (2021) employed healthy and inflamed triple cell model of the intestines to investigate the toxicity described above of polystyrene (agglomerates approximately 225 nm) and polyvinyl chloride (agglomerates approximately 1150 nm) micro/nanomaterials. Following 21 days to establish the system, cells were exposed to a single administration of the materials at concentrations of 1, 5, 10 or 50 µg/cm². No acute NM-induced effects in terms of cell death, DNA damage or inflammation in the healthy intestine model was found. However, in the inflamed intestine model the polystyrene materials resulted in an increased secretion of IL-1β and loss of epithelial cell integrity in a concentration dependent manner (Busch et al. 2021).

NM induced toxicity in pathophysiological hepatic models

In a unique study, the hepatic toxicity following iv exposure to Ag NM (approximately 30 nm) was assessed in models of alcoholic liver disease in vitro and in vivo. In these experiments, Kermanizadeh et al. (2017) randomly divided 8-week-old female C57BL6 mice into two groups with one fed an all-liquid diet for 25 days while the other group received an all-liquid diet supplemented with 5% ethanol. Following the initiation of disease, mice were injected with either 25 or 100 µg NMs for 24 or 168 hr. Kermanizadeh et al. (2017) showed that NM-induced hepatic health effects were significantly enhanced in the alcohol fed mice in comparison to controls in terms of an organ specific inflammatory responses, metabolic and toxicological changes in blood biochemistry, acute phase response and hepatic pathology as evidenced by marked changes in intrahepatic architecture, granuloma formation in different zones of the liver, hepatocyte necrosis, parenchymatous degeneration, vacuolar generation, and the destruction of the liver plates. Further, and extremely importantly, the disease state significantly influenced and hindered organ ability for recovery following NM exposure. In the same study, in vitro findings demonstrated ethanol pre-treatment of HepG2 cells (human hepatoma cell line) resulted in an enhanced IL8 response post material NM exposure (Kermanizadeh et al. 2017).

In an interesting and comprehensive study, the impact of co-exposure of two ZnO NMs (approximately14 and 60 nm) and lead (Pb) was administered to 5-week-old male C57BL6 mice on normal or high fat diets (Jia et al. 2017). Mice were orally administered NMs (200 mg/kg) or NMs/Pb (200 and 150 mg/kg for NMs and Pb respectively) daily for a period of two weeks. Jia et al. (2017) quantified individual NMs in the liver which was similar in normal and high fat animals. However, co-exposure of NMs increased the rate of deposition of Pb in the liver by 2-fold compared to levels following Pb alone. Subsequently, the investigators noted that ZnO NMs or ZnO/Pb exposure of normal diet mice only resulted in minimal pathological damage. However, the same exposures in the high fat diet mice induced significant liver damage. In addition, the high fat diet resulted in pathological alteration including steatosis, spotty cell necrosis and mild vacuolar degeneration. Further, a reduction in the SOD activity and rise in malondialdehyde content was found only in the high fat diet animals following co-treatment of NMs. Data clearly showed that hepatic toxicity attributed to NMs or co-administrations was markedly amplified by pre-existing disease (Jia et al. 2017).

In another study, a mouse model of inflammatory bowel disease (IBD) (indomethacin mediated) was utilized to investigate the adverse effects of ZnO NMs on the liver. In these experiments, (Du et al. 2018) exposed 8-week-old male ICR mice were to two differently sized NMs (20 nm×100 nm and ~200 nm) at a single oral dose of 1 g/kg for up to 24 hr. Higher quantities of Zn were detectable in the livers of IBD mice compared to healthy animals. Evidence thus indicates the importance of the health status of animals on the bio-distribution of NMs with the implication that intestinal injury/increased inflammatory state plays a critical role in hepatic NM distribution in mice which was most apparent for the 200 nm NMs

(Du et al. 2018).

Another 2018 study aimed to assess the therapeutic effects of Au NMs (approximately 8 nm) in male Wistar rats (300 g) following alcohol and methamphetamine induced liver injury. The animals were administered orally three daily doses of the NMs (181.48, 362.48 or 724.96 μg/kg) for one hr prior to treatment with ethanol for 28 days. de Carvalho et al. (2018) reported that significantly greater myeloperoxidase activity was detected in damaged livers compared to controls, which was attenuated in animals treated with the highest dose of Au NMs. Further, Au treatment increased glutathione (GSH) levels in the organ. Treatment with ethanol and methamphetamine resulted in enhanced inflammatory responses as noted by increased levels of IL-1β and TNF-α. Interestingly, combined treatment of ethanol, methamphetamine and the highest dose of the NMs was associated with elevated IL10 (potent anti-inflammatory) hepatic response. Histopathological analysis of the diseased rat livers exhibited steatosis, lymphocyte and neutrophil infiltration and areas of necrosis. However, administration of Au NMs reduced histopathological damage described above in. Finally, quantification of NF-κB, F4/80, protein kinase B, phosphatidylinositol-4,5-bisphosphate 3-kinase, pro collagen III, allograft inflammatory factor 1, extracellular signal-regulated kinases 1/2, transforming growth factor-β, fibroblast growth factor, superoxide dismutase 1 and glutathione peroxidase 1 genes showed that NM treatment might reduce the activation of Kupffer cells (KC)s and hepatic stellate cells hence diminishing inflammation, fibrosis and oxidative stress in the organ (de Carvalho et al. 2018).

The effects of Au NM (approximately 25 nm) exposure in mice with pre-existing obesity was investigated. In these experiments 18-week-old male C57BL6 mice on a high fat diet were exposed to the NMs via the ip route at doses of 0.785 or 7.85 μg/g/day for a further 5 weeks. Chen et al. (2018) that in the high dose Au NM exposed animals there was improved glycemic control and reduced blood lipid levels. In addition, in the liver of Au NM treated mice, there was a downregulation of inflammatory markers TNF-α and toll like receptor 4 and improved lipid metabolic markers FOXO1, GLUT-4, ATGL and CPT-1α. Taken together data suggest that Au NMs improved glucose and fat metabolism in mice with long-term obesity (Chen et al. 2018).

In a 2022 in vitro investigation, two disease initiation and maintenance protocols were developed, described and used to mimic steatosis and pre-fibrotic non-alcoholic steatohepatitis (NASH) states in scaffold-free 3D liver tissues composed of primary human hepatocytes, hepatic stellate cells, KCs and sinusoidal endothelial cells. The physiological and pathophysiological tissues were used for comprehensive toxicological assessment of a panel of NMs which included approximately 150 nm ZnO NMs, approximately 50 nm, CeO₂ NMs and food grade TiO₂ (E171). In these trials the diseased microtissues were exposed to the materials at very low repeated doses (highest dose of 10 µg/ml) for a period of two weeks. (Kermanizadeh et al. (2022) demonstrated significant evidence that pre-existing liver disease was extremely important in intensification of xenobiotic-induced hepatotoxicity as manifested in cell death, inflammation and histopathology. Data showed that NMs were able to fully activate hepatic stellate cells. Therefore, evidence suggests that it is crucial that all stages of the wide spectrum of liver disease are incorporated in xenobiotic hazard assessment strategies (Kermanizadeh et al. 2022b)

NM induced toxicity in pathophysiological central nervous system models

In an interesting study, the toxicological impact of CeO₂ NMs (approximately 5 nm) exposure was investigated in mouse models of Alzheimer’s and vascular disease. Wahle et al. 2020) utilized female 5XFAD transgenic mice (8–11-week-old) as a model to simulate major features of Alzheimer’s disease amyloid pathology, while 10–12-week-old female ApoE^−/− mice were used as a model of vascular disease. Mice were exposed via nose-only inhalation to CeO₂ NMs with increasing quantities of zirconium (0%, 27% or 78% Zr) or clean air over a 4-week period (4 mg/m³ for 3 hr/day, 5 days/ week). The effect of exposure to NMs on the behavior of mice was determined using two tests – namely the string suspension and the X-maze test. In the 5xFAD and ApoE^−/− models, NMs did not markedly affect X-maze scores. However, following inhalation exposure to the 78% Zr-doped NMs, changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE^−/− and 5xFAD mice, respectively. However, no significant treatment-related changes in neuro-inflammation (amount of Iba-1 positive microglia cells and astrocyte marker glial fibrillary acidic protein assessed via immunohistochemical analysis) and oxidative stress (level of Nrf2 or HO-1) were observed. Data is suggestive of the fact that inhalation exposure to NMs selected did not aggravate CeO₂ Alzheimer’s phenotype in the mice model (Wahle et al. 2020).

Hullmann et al. (2017) investigated the toxicological consequences of long-term exposure to diesel engine exhaust on models of Alzheimer’s disease. In this study, female 5xFAD mice were exposed in whole body inhalation chambers for 3 or 13 weeks to diesel engine exhaust (0.95 mg/m³, 6 hr/day, 5 days/week). Hullmann et al. (2017) used Y and X-maze tasks to examine spatial working memory in mice in association with the diesel exhaust. Results from these tests showed no memory deficit attributed to exhaust exposure. Subsequently, to determine the impact of the NMs on plaque formation in the disease model parasagittal brain sections were labelled with an Aβ42 antibody. The immunohistology illustrated higher cortical Aβ plaques three weeks post diesel exhaust exposure. These finding were further corroborated by observing higher levels of Aβ42 in exposed brain homogenates (Hullmann et al. 2017).

Sofranko et al. (2022) employed 9-week-old female 5xFAD transgenic mice as a model selected for assessment of sub-chronic oral exposure to SiO₂ (approximately 13 nm) and CeO₂ (approximately 50 nm) NMs on Alzheimer’s disease pathology. The animals were fed ad libitum for 3 or 14 weeks with pellets loaded with 0.1% or 1% (w/w) of the NMs or appropriate controls. Data showed that the long-term oral exposure of either material did not markedly affect behavior (X-maze, balance beams and open field test), histopathology, plaque formation acceleration (Aβ42 staining), Aβ40 and Aβ42 protein levels, or oxidative stress (tissue glutathione levels). Overall, findings indicate that long-term oral exposure to the NMs utilized within this investigation exerted no significant neurotoxic and Alzheimer’s disease promoting effects (Sofranko et al. 2022).

A summary of all the studies highlighted in the main body of this review can be found in Tables 1-5.

Table 1. Summary of NM-induced adverse effects in pathophysiological pulmonary models.

Model of disease	NMs and size	Route of exposure of NMs (where applicable)	NM dosing regimen	Was NM-induced toxicity augmented in pathophysiological (pre-existing disease) models as compared to healthy models?	Reference
Ovalbumin sensitised 8-week-old Wistar rats	SiO2 – ~ 20 nm	IT	30 daily doses of 4 or 8 µg	Yes	Han et al. 2011
House mite mediated allergy 10–16 week-old C57BL6 mice	MWCNT – diameter ~ 30 nm and length 5–15 µm	IT	Single dose 2 mg/kg	Yes	Carvalho et al. 2018
LPS mediated inflammation 7-9-week-old Sprague-Dawley rats	MWCNT – diameter – 10-30 nm and length 0.3–50 mm	IT	Single dose 4 mg/kg	Yes	Cesta et al. 2010
LPS mediated inflammation BALB/c mice	TiO2 – ~ 20 nm	IP	Single dose 40 mg/kg	Yes	Moon et al. 2010
Ovalbumin sensitised 9-week-old mice (species not specified)	TiO2 – size not stated	Inhalation (inhalation chamber)	50 µg/m^3 for 2 hr per day for three consecutive days	Yes	Kim et al. 2017
Ovalbumin sensitised 12-week-old BALB/c mice	TiO2 – ~ 170 nm	IP	Four consecutive daily doses of 100 µg	Yes	Mishra et al. 2016
Ovalbumin sensitised 8-week-old BALB/c mice	TiO2 – < 5 µm or 50 nm	Inhalation (inhalation chamber)	10 mg/m^3 for 2 hr per day three times a week for four weeks	No	Rossi et al. 2010
Ovalbumin sensitised 12-week-old Wistar rats	SiO2 – 10-20 nm	IT	4 or 8 µg for 30 consecutive days	Yes	Han et al. 2011
Ovalbumin sensitised 11-week-old BALB/c mice	SiO2 – 30, 70, 300 or 1000 nm	Intranasal	Single dose of 250 µg	Yes	Yoshida et al. 2011
Ovalbumin sensitised 8-week-old BALB/c mice	SiO2 – ~ 90 nm	Intranasal	Four doses over 8 days – 400 µg/animal	Yes	Brandenberger et al. 2013
Ovalbumin sensitised 6-week-old BALB/c mice	SiO2 – ~ 100 or 400 nm	Intranasal	6 doses over 14 days 10 mg/kg	Yes	Han et al. 2016
Ovalbumin sensitised 8-week-old BALB/c mice	Ag – ~ 30 nm	Inhalation (inhalation chamber)	3.3 mg/m^3 for 6 hr per days on seven consecutive days	No	Chuang et al. 2013
Ovalbumin sensitised 6-8-week-old BALB/c mice	Au – 5 nm	Intranasal	2 doses on day 1 and 7 (dose not stated)	Yes	Omlor et al. 2017
Ovalbumin sensitised 7-week-old BALB/c mice	Fe2O3 – ~ 35 or 150 nm	IT	4 administrations over 12 hr at doses of 100, 250 or 500 µg/animal	Yes	Ban et al. 2013
Ovalbumin sensitised 7-week-old BALB/c mice	ZnO – ~ 50 nm	Oropharyngeal aspiration	2 administrations on day 1 and 7 at doses of 1 or 5 mg/kg	No	Huang et al. 2015
Ovalbumin sensitised 6-week-old ICR mice	Carbon black – 15 or 55 nm	IT	Once a week for 6 weeks	No	Koike et al. 2008
Ovalbumin sensitised 8-week-old BALB/c mice	Carbon black – 15 nm	Pharyngeal aspiration	4 administrations over 9 days at doses of 2.5 mg/kg	Yes	Kroker et al. 2015
Ovalbumin sensitised 6-8-week-old C57BL6 mice	MWCNT – length 0.5–40 µm	Nose only inhalation	100 mg/m^3 for 6 hr	Yes	Ryman-Rasmussen et al. 2009
Ovalbumin sensitised 7-week-old ICR mice	MWCNT – ~ 65 nm, length not specified	IT	50 µg/animal once a week for 6 weeks	Yes	Inoue et al. 2009
Ovalbumin sensitised 7-week-old ICR mice	MWCNT – diameter ~ 15 nm, length 0.1–10 µm	Intranasal	4 doses over 16 days – 30 µg/animal per dose	Yes	Mizutani, Nabe, and Yoshino 2012
House dust mite and diesel exhaust mediated sensitisation 6-8-week-old BALB/c mice	CeO2 – 25 nm	Intranasal	19 doses over 19 days 2.5 or 75 µg/kg	Yes	Meldrum et al. 2020
House dust mite mediated sensitisation 6-8-week-old BALB/c mice	CeO2 – 25 nm	Intranasal	19 doses over 19 days 2.5 or 75 µg/kg	Yes	Meldrum et al. 2018
Ovalbumin sensitised 6-week-old BALB/c mice	SiO2 – 50 nm	Intranasal	3 administrations over 5 days at doses of 5, 10 or 20 mg/kg	Yes	Ko et al. 2020
Ovalbumin sensitised 8-week-old BALB/c mice	SiO2 – ~ 100 nm	Intranasal	200 µg over 3 consecutive days	Yes	Park et al. 2015
Ovalbumin sensitised 6-week-old BALB/c mice	TiO2 – ~ 50 nm	Inhalation (inhalation chamber)	200 µg/m^3 for 2 hr	Yes	Kim et al. 2020
Respiratory syncytial virus 6-8-week-old C57BL6 mice	TiO2 – ~ 100 nm	IT	Single dose 10–60 µg/animal	Yes	Smallcombe et al. 2020
Primary human epithelial cells from lungs of paediatric patients infected with syncytial virus (compared to not infected cells)	TiO2 – ~ 100 nm	NA	10–100 µg/cm^2	Yes	Smallcombe et al. 2020
Ovalbumin sensitised primary leukocytes (compared to non-sensitised cells)	Carbon black – ~ 20 or 40 nm	NA	12.5 µg/ml	Yes	Lefebvre et al. 2014
Primary lymphocytes from asthma patient (compared to cells from healthy individuals)	ZnO – ~ 150 nm	NA	10–40 µg/ml	No	Kumar et al. 2015

Discussion

The field of nanoscience and nanotechnology is constantly evolving with ever increasing number of products incorporating and utilizing nano-scaled materials. Despite their numerous advantages, two decades of extensive nanotoxicological research has clearly demonstrated the importance of assessing the potential associated risks of exposure to a range of NMs. To fully exploit the potential NMs in fields of nanotechnology and nanomedicine it is imperative that any safety concerns are fully addressed. An important consideration in this, is the inclusion of the absolute huge numbers of the general population with either diagnosed or sub-clinical symptoms of a range of chronic diseases plaguing the populace in future hazard and risk assessment strategies for NMs. To date, the main body of work in the field of nanotoxicology focussed on test models representing healthy individuals. Of course, this was and remains, an absolute necessity. However, with nanotoxicology moving away from its infancy and the burden of chronic disease increasing globally both in terms of morbidity and mortality it is critical that risk assessment strategies also evolve and align with the requirements of the general population who are exposed to such materials on a day-to-day basis. In this review the critical studies that were carried out in the field to assess the NM-induced health effects in models were presented which are representative of individuals with pre-existing medical conditions.

The interaction of NMs with biological surroundings is governed by a range of material properties including but not limited to size, composition shape, bio-persistence, charge and coating (Sukhanova et al. 2018). These factors affect the internalization and uptake of the materials and subsequently their potential toxicity which may manifest as cytotoxicity, inflammation, autophagy, carcinogenicity and genotoxicity (Verdon et al. 2022). During cellular uptake certain NMs agglomerate, resulting in size-dependent cellular internalizing pathways taken up by these compounds (Lunnoo, Assawakhajornsak, and Puangmali 2019). A significant and ever-increasing body of toxicology data is now available for NMs which employed a wide variety of test systems, protocols and toxicological end-points. It is abundantly clear that all NMs are not equally toxic and these disparities are, to a large extent, based upon physicochemical properties as well as varying experimental protocols, exposures and dosing strategies. Finally, the literature clearly shows that in in vivo studies, the route of exposure is vital in the proportion of the NM dose reaching various organs and hence significantly influences potential toxicity.

As stated above, chronic diseases account for an estimated 7 out of 10 deaths globally every year. This infers that a significant proportion of global population numbering in the billions are currently residing with a range of long-term health conditions including asthma, chronic liver disease and cardiovascular complications amongst many more.

With an aging global population, chronic respiratory diseases are becoming a prominent cause of death and disability. This trend has been increasing for over a decade with the recent COVID-19 pandemic likely to add to this further (Ekezie et al. 2021). Among the range of respiratory diseases, COPD and asthma have the highest prevalence. In 2017, there were 3.2 million deaths attributed to COPD and 495,000 deaths to asthma. In the same year, the incident cases of chronic respiratory diseases were estimated at 300 million mostly due to asthma (69%) and COPD (29%) (WHO 2022). Asthma is categorized by airway hyperresponsiveness, inflammation and overproduction of mucus all of which contribute to the obstruction of the airways resulting in characteristic symptoms associated with the disease (Krishnan et al. 2012). Although different variants of asthma have been identified allergic asthma is the most well investigated. The current understanding of the allergic variant of disease is uptake of the allergen by pulmonary dendritic cells (specialized antigen presenting cells), which subsequently process and present the antigen to T helper cells. This results in activation of different naive lymphocytes in Th2 type cells. Th2 cells then stimulate humoral immune response promoting B cell proliferation and production of IL4 and IL13. The activated B lymphocytes produce antigen specific IgE, which binds to the allergen and receptors on mast cells resulting in their degranulation and release of histamine, leukotrienes leading to further inflammation. Allergic asthma is also characterized by an influx of innate immune cells in response to the allergen amongst which eosinophils, macrophages and neutrophil are the most important (Leblond et al. 2009). Finally, epithelial cells (inflammatory cytokine secretion), smooth muscle cells (hypertrophy and hyperplasia) and fibroblasts (collagen deposition) are all involved in progression of disease and symptoms (Kudo, Ishigatsubo, and Aoki 2013)

Not surprisingly, the scrutiny of the literature depicts nanotoxicological studies using pathophysiological pulmonary test models significantly outnumbering the rest of the biological systems highlighted in this review such as cardiovascular, hepatic, gastrointestinal and central nervous. This trend is also true for nanotoxicological studies in general and understandable since inhalation exposure is the primary and most important route of exposure for NMs. Investigators reported that OVA sensitized asthmatic mice models are the most widely used pathological model to investigate NM-induced toxicity with the exception of (Carvalho et al. 2018); (Cesta et al. 2010; Meldrum et al. 2018, 2020; Moon et al. 2010). NMs are capable of modulating the asthmatic inflammatory response with the material-induced toxicological outcomes as evidenced by cell counts in BALF, pro-inflammatory cytokine levels and serum IgE quantities (Janssens et al. 2012). Data indicate that in almost all cases NM-mediated toxicity was enhanced in the disease models compared to healthy counterparts (augmentation of disease state overall).

Cardiovascular diseases are the leading causes of morbidity and mortality globally with a of range of conditions accounting for an estimated 32% of all world-wide deaths in 2019 (WHO 2022). In the UK alone it is estimated that almost 8 million individuals are currently living with heart and circulatory diseases (British Heart Foundation 2022). Cardiovascular related diseases include, but are not limited to, atherosclerosis, heart failure, cardiac arrhythmia, vasculopathy, hypertension and myocardial ischemia-reperfusion injury. Oxidative stress is believed to be extremely important in the development and evolution of cardiovascular diseases (Pignatelli et al. 2018; Shkirkova et al. 2020; Wang and Kang 2020). Considering oxidative stress is one of the main mechanisms of NM-induced toxicity, the significance of NMs in individuals with pre-existing cardiovascular disease should not be ignored.

The review of existing literature showed that there were relatively few studies that investigated NM-induced toxicity in pre-existing cardiovascular disease test models (although hundreds of studies on nanomedicines or surface-modified metal particles were identified). This was rather surprising considering the importance of cardiovascular disease and its prevalence in the general population. Although it is speculation, there may be a few possible explanations for this. Firstly, due to the varied range of manifestations of vascular and cardiovascular diseases, the test models are limited to be more representative of late-stage cardiovascular disease and not suited for fully capturing NM-induced toxicity. It might also be argued that generally cardiovascular disease is often diagnosed late so presence of NMs (and their potential toxicity) in someone with serious health complications might not necessarily be that important in the larger picture of the individual’s health status and eventual prognosis. That being said, all 5 studies highlighted in the main section noted an augmentation of NM-induced adverse effects in the pre-existing disease models as compared to healthy controls.

Gastrointestinal disease affects the esophagus, stomach, intestines, rectum as well as the liver, gall bladder and pancreas. In that sense, our use of sub-heading “gastrointestinal tract” is not entirely accurate. The focus on the review for the “gastrointestinal tract” was solely the intestines. The most prevalent intestinal disease currently affecting the general population is inflammatory bowel disease (IBD) which is a term used to describe Crohn’s disease and ulcerative colitis. It is believed that the prevalence of IBD in 2016 was 142 out of every 10,000 adults. In addition, the prevalence increased between 2006 and 2016 by 34% (Freeman et al. 2021). Crohn’s disease might initiate transmural inflammation and potentially affect any part of the GIT (most commonly, the terminal ileum or the perianal region). Crohn’s disease is often associated with complications such as abscesses, fistulas and strictures (Maharshak et al. 2008). In contrast, ulcerative colitis is typified by mucosal inflammation and limited to the colon (Lai et al. 2021). Despite the unknown etiology of IBD, it is generally accepted that progressive inflammation is key in initiation and exacerbation of disease conditions. Considering the fact that the intentional or accidental ingestion of NMs is one of the most important routes of exposure for NMs, it is conceivable that the intestines were the primary focus of investigations in nanotoxicological studies. Examination of published literature showed that numerous studies determined NM-induced effects in models representative of healthy individuals in general populations, yet to the best of our knowledge only two measured particle toxicity in models which represent the diseased gut (Busch et al. 2021; Kämpfer et al. 2021). Due to the small number of studies, it is difficult to form any meaningful conclusions on the significance of pre-existing intestinal disease in NM-induced toxicity. However, since a defective mucosal barrier “leaky gut” is a main characteristic of IBD, (Michielan and D’Inca 2015) it seems logical that this might result in increased translocation of particulates into systemic circulation and subsequent adverse effects in humans. Data suggest that assessment of NM-mediated toxicity in models that are representative of diseased intestines in humans should be further explored.

Non-alcoholic fatty liver disease (NAFLD) is a general term encompassing the hepatic manifestation of metabolic syndrome and refers to a wide continuum of liver disorders (Kermanizadeh, Powell, and Stone 2020b). NAFLD might progress to more serious conditions such as Non-alcoholic steatohepatitis (NASH) characterized by hepatic inflammation and damage. Recently, NAFLD was termed metabolic associated fatty liver disease (MAFLD) as it describes its etiology more precisely (Tilg and Effenberger 2020). It is understood that liver disease is governed and progressed by a pro-inflammatory state in the organ. Chronic inflammation and subsequent repair in the organ might ultimately lead to severe damage displayed as fibrosis and eventual cirrhosis which potentially progresses to hepatocellular carcinoma (Ioannou et al. 2020; Rodriguez-Antonio et al. 2021). Liver disease is also intimately associated with three of the major global health challenges: obesity, metabolic syndrome and diabetes. Globally in 2022, NAFLD has extremely high prevalence. It is believed the condition affects up to 40% of the adult population, among whom nearly 30% progress to NASH. Further, it is now estimated that 2 out of every 3 adults in the UK have early stages of NAFLD. Mortality rates for liver disease have increased 400% since 1970, and in individuals aged under 65 have risen by almost 500% (British Liver Trust 2022). In addition, approximately 75% of patients who die due to cirrhosis are currently unaware that they have liver disease and the majority have entirely normal liver serum biochemistry until they present to hospital as an emergency. This important and additional complication is clinically significant as large numbers of individuals globally suffer from a spectrum of sub-clinical liver diseases without any apparent visible manifestations which often remain clinically asymptomatic and undetected.

The liver is of upmost importance in a nanotoxicological context, as this organ was found to accumulate NMs at high volumes compared with other organs following the major routes of exposure, such as inhalation (Kobos et al. 2020; Sadauskas et al. 2009). In addition, with the emergence and ever-increasing advancements in the field of nanomedicine, the intentional administration of particulates in a range of nano-formulations into systemic circulation is routine such that an understanding of hepatic particle toxicology is crucial. As demonstrated in the main section of the review, the relevant literature clearly demonstrates that NM-induced adverse effects in the organ are significantly enhanced in the diseased state. Further, the pre-existing disease might influence the liver’s ability for regeneration post-NM challenge. Therefore, it is critical that a range of liver diseases be considered for inclusion in future hazard assessment strategies for xenobiotics. This is of upmost importance for mild disease in the liver (steatosis) since the adult population that are residing with undiagnosed mild liver conditions is significant and numbers in the billions.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases affecting millions worldwide. In 2020, in the United States, over 6 million are understood to be living with AD (Alzheimer’s Association 2022) while in the same year an estimated one million individuals were reported to be affected by PD (Parkinson’s Foundation 2022). Alzheimer’s disease is clinically manifested as progressive impairment in cognition, learning ability, loss of memory function and reasoning (Jiang, Sun, and Chen 2016). On the other hand, PD is characterized by impairment of motor symptoms as well as postural instability at a more advanced stage (Hess and Hallett 2017). Amongst numerous risk factors for both neurodegenerative conditions, increasing age of patients is very important. In our review of the available literature, only three studies that used pathophysiological models representative of neurodegenerative disease in humans were identified. Due to the small number of studies, it is difficult to form any meaningful conclusions on the significance of pre-existing neurodegenerative disease in NM-induced toxicity, although it appears that the NMs themselves and their inherent toxicity seem to be important (this is further discussed below). An additional and important consideration when analyzing the published data is whether the length of the experiments following NM exposures were sufficient for disease to fully manifest and therefore be detected. As an example, in central nervous system early symptoms such as inflammation might occur in advance of cognition impairment which might not necessarily be detected unless the length of the experiment was sufficiently prolonged.

An important issue that needs attention but will not be covered in detail in this review (as it does not fall within its remit) is the availability, suitability and costs of the pathophysiological models for nanotoxicological investigations. Briefly, out with a few well-established in vivo models, the customization and reproducibility of numerous pathophysiological models is still far from ideal. This is most notable for the advanced in vitro models of disease. This standardization and acceptance of these test systems is an absolute must for any regulatory approval. On a similar theme, the literature clearly shows that pathophysiological spheroid, microfluidics platforms and 3D bioprinting platforms are under-utilized in the nanotoxicological context despite being more predominant in other fields. A potential explanation for this might be the extremely high cost and technical challenges of working with some of this technology. Finally, a limiting step in incorporation of pathophysiological models in toxicity testing is the clear lack of availability of appropriate test systems (in vivo, in vitro and ex vivo) that adequately and accurately represent and replicate disease in humans. The importance of this issue is discussed in detail elsewhere (Tutty, Movia, and Prina-Mello 2022; Van Norman 2019).

As mentioned earlier, cancer was not included in this review. There were a number of reasons for this. Firstly, due to the varied range of cancers affecting different body systems and organs and often different cancers affecting the same organ/system, it is almost impossible to include these in testing strategies. Most importantly cancers differ (1) in their aggressiveness, (2) are considered as life-threatening disease and dependent upon type and stage, and (3) are not necessarily chronic conditions. To this end, from a risk assessment perspective, it can be argued that NM-mediated effects exert little importance in individuals who have been diagnosed with life-threatening (and on occasion terminal) cancer. That being said, and it might be obvious, the above statement only applies to established disease states (cancer) and not to certain NMs’ potential genotoxic and carcinogenic properties that might progress due to development of cancer. Finally, due to their relative low cost, accessibility, ease of use and suitability for high throughput analysis, cancer derived human cell lines are often used in vitro toxicological studies and have been for decades. However, the majority of these models are representative of healthy individuals in the general population. The merits and pitfalls to this approach are discussed in detail elsewhere (Pamies and Hartung 2017; Wilding and Bodmer 2014).

The comprehensive scrutiny of the literature above clearly showed that a large majority of studies indicated that pre-existing disease state is crucial in the augmentation of NM-mediated toxicological consequences and, as a result, progressing disease state leads to more serious conditions. In this review, only a few studies were identified in which NM toxicity observations were not enhanced in the diseased states (Chuang et al. 2013; de Carvalho et al. 2018; Kämpfer et al. 2021; Koike et al. 2008; Kumar et al. 2015; Rossi et al. 2010; Sofranko et al. 2022; Wahle et al. 2020). These NMs differ in their inherent toxicity due to varying physicochemical properties. Although no meaningful pattern was detectable in terms of certain material type and their toxicity on pre-existing disease, it might be worth mentioning that two focused on TiO₂, three on CeO₂ NMs. From review of the literature and our own studies on the topic (with regards to the hepatic and pulmonary systems), it was suggested to incorporate pre-existing disease models in the nanotoxicological context. This is particularly critical for chronic sub-clinical conditions that affect billions in the general population such as asthma and steatosis. This approach is probably less important and not needed in severe and life-threatening conditions where NMs and their potential adverse effects have less significance to the overall health of the individual. Of note, the literature also demonstrated the need for more epidemiological studies investigating NMs’ adverse effects in humans.

In the field of toxicology, there is a principal prerequisite for the generation of meaningful and reliable data, which is reflective and relevant to the population that might encounter the potential toxin, ultimately supporting risk assessment, product development and associated technologies. This statement is relevant for NMs and advancements in the field of nanotechnology. In order to carry out a well-informed, evidence-based risk assessment for the emerging NMs on the market, a comprehensive understanding of NM-initiated risks is required. A critical risk assessment requires knowledge regarding the level of exposure to the manufactured NM, route of exposure, the bio-persistence and accumulation in the organism and inherent toxicity of the material in question. In order to generate robust data for the latter (inherent toxicity of the material) coupled with the fact that significant proportion of global populace numbering in the billions are living with a range of chronic undiagnosed health conditions, it is recommended that NM toxicology in certain pre-existing diseases is incorporated and considered for hazard assessment.

Table 2. Summary of NM-induced adverse effects in cardiovascular disease models.

Model of disease	NMs and size	Route of exposure of NMs (where applicable)	NM dosing regimen	Was NM-induced toxicity augmented in pathophysiological (pre-existing disease) models as compared to healthy models?	Reference
Hyperlipidaemic model of atherosclerosis 12-week-old C57BL6 ApoE^−/− or LDL receptor KO mice on western diet	Supermagnetic iron oxides – ~120-180 or ~ 30 nm	IV	1 mg/kg	Yes	Segers et al. 2022
LPS spike 10-week-old C57BL6 ApoE^−/− mice	Carbon black- ~ 20 nm	IT	Two doses of 25.6 µg/animal per week for 10 weeks	Yes	Christophersen et al. 2016
Model of hypertension -angiotensin II exposed 10-week-old BALB/c	Au NMs – ~ 40 nm	IT	4 doses of 0.5 mg/kg over 28 days	Yes	Ferdous et al. 2022
Cardiac hypertrophy Fertilized egg with 2,2-azobis(2-amidinopropane) dihydrochloride	ZnO NMs – ~ 100 nm	NA	10–40 µg/egg	Yes	Nagarajan et al. 2022
LPS spike HUVECs (as compared to non LPS spiked HUVECs)	Ay nanorods – ~ 50 nm, diameter ~ 25 nm	NA	10 µg/ml	Yes	Li et al. 2019

Table 3. Summary of NM-induced adverse pathophysiological gastrointestinal models.

Model of disease	NMs and size	Route of exposure of NMs (where applicable)	NM dosing regimen	Was NM-induced toxicity augmented in pathophysiological (pre-existing disease) models as compared to healthy models?	Reference
IFN-Ƴ model of inflamed intestine composed if epithelial cells, goblet cells and macrophages (as compared to non-primed cells)	Ag – ~ 250 nm TiO2 – ~ 300 nm	NA	1 mg/kg	No	Kämpfer et al. 2021
IFN-Ƴ model of inflamed intestine composed if epithelial cells, goblet cells and macrophages (as compared to non-primed cells)	Polystyrene NM – ~ 225 nm Polyvinylchloride ~ 1150 nm	NA	Up to 50 µg/cm^2	Yes	Busch et al. 2021

Table 4. Summary of NM-induced adverse pathophysiological hepatic models.

Model of disease	NMs and size	Route of exposure of NMs (where applicable)	NM dosing regimen	Was NM-induced toxicity augmented in pathophysiological (pre-existing disease) models as compared to healthy models?	Reference
Alcohol-mediate disease 8-week-old C57BL6 mice	Ag – ~ 30 nm	IV	25 or 100 µg/animal	Yes	Kermanizadeh et al. 2017
High fat induced steatosis 5-week-old C57BL6 mice	ZnO – ~ 15 or 60 nm	Oral	150 mg/kg daily for 2 weeks	Yes	Jia et al. 2017
Indomethacin induced inflammatory bowel disease 8-week-old ICR mice	ZnO – 20 × 100 nm or ~ 200 nm	Oral	1 g/kg	Yes	Du et al. 2018
Alcohol and methamphetamine induced liver injury 300 g Wistar rats	Au nm – ~ 10 nm	Oral	Three daily doses of up to 7.24 µg/kg	No	de Carvalho et al. 2018
High fat induced steatosis 18-week-old C57BL6 mice	Au nm – ~ 25 nm	IP	0.785 or 7.85 µg/day for five weeks	No	Chen et al. 2018
LD and LPs induced steatosis and NASH models 3D multi-cellular model composed of primary hepatocytes, Kupffer cells, endothelial cells and stellate cells (as compared to healthy liver model composed of cell populations	ZnO – ~ 150 nm CeO2 – ~ 50 nm Food grade TiO2	NA	seven doses over 2 week period (up to 10 µg/ml)	Yes	Kermanizadeh et al. 2022

Table 5. Summary of NM-induced adverse pathophysiological central nervous system models.

Model of disease	NMs and size	Route of exposure of NMs (where applicable)	NM dosing regimen	Was NM-induced toxicity augmented in pathophysiological (pre-existing disease) models as compared to healthy models?	Reference
Model of Alzheimer’s disease 8–11-week-old 5XFALD transgenic mice	CeO2 with varying quantities of zirconium – ~ 5 nm	Inhalation (nose-only)	4 mg/m^3 for 3 hr/day, 5 days a week for 4 weeks	No	Wahle et al. 2020
Model of Alzheimer’s disease 8–11-week-old 5XFALD transgenic mice	Diesel engine exhaust	Inhalation (whole body chamber)	0.95 mg/m3, 6 hr/day, 5 days/weeks for 3 or 13 weeks	Yes	Hullmann et al. 2017
Model of Alzheimer’s disease 8–11-week-old 5XFALD transgenic mice	SiO2 – ~ 15 nm CeO2 – ~ 50 nm	Oral	0.1 or 1% w/w in feed pellet for 3 or 14 weeks	No	Sofranko et al. 2022

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