Potential Toxicity of Nanoparticles for the Oral Delivery of Therapeutics

Abstract

Nanoparticles (NPs) offer a promising solution for orally delivering therapeutic substances due to their capability to surpass traditional drug delivery system (DDS) limitations like low solubility, bioavailability, and stability. However, the possible toxic effects of using NPs for oral therapeutic delivery raise significant concerns, as they might interact with biological systems unexpectedly. This chapter aims to comprehensively understand the potential toxicity of NPs employed in oral therapeutic delivery. Factors such as size, surface area, surface charge, and surface chemistry of NPs can impact their toxicity levels. Both in vitro and in vivo models have been utilised to evaluate NPs toxicity, with in vivo models being more suitable for anticipating human toxicity. The possible toxic consequences of different NPs varieties, including polymer, lipid, and metal NPs, have been documented. Ultimately, grasping the potential toxicity of NPs in oral therapeutic delivery is essential for creating safe and effective DDS.

1. Introduction

The use of nanoparticles (NPs) for oral drug delivery is a ground-breaking and rapidly growing area of research [1]. NPs are solid colloidal drug delivery systems (DDS) with sizes ranging from 1 nm to 1000 nm. They comprise polymers, lipids, carbon, silica, or metal encapsulating the drug moiety [2, 3, 4, 5]. Encapsulating drugs within NPs can increase stability, solubility, and bioavailability, ultimately boosting their therapeutic effectiveness [6]. Recent advances in biomedical research have led to the successful enhancement of therapeutic agents for treating various diseases. However, a significant challenge remains in efficiently delivering these agents to the target site [7]. NPs can be engineered with specific characteristics to optimise the DDS [4]. For example, biocompatible polymer NPs are coated to enhance their presence in the bloodstream or be equipped with unique ligands or antibodies that focus on specific target cells or tissues [2]. By directing drug delivery to specific locations within the body, NPs can reduce negative side effects and increase the effectiveness of treatments [8].

Oral drug administration is the most prevalent method and the favoured option for patients, as it is non-invasive, user-friendly, and well-accepted [9]. However, traditional forms like tablets and capsules may have a rapid and inadequately regulated drug release, potentially leading to drug degradation and alteration due to the gastrointestinal tract (GIT) environment (like, changes in pH, digestive enzymes, and microbiota) [10]. NPs can address these issues by safeguarding drugs from degradation and promoting their absorption through the intestinal barrier. Moreover, oral drugs can be directed to specific areas within the GIT to provide localised treatment for stomach and colorectal cancers, infections, inflammation, bowel disorders, gastro-duodenal ulcers, and gastroesophageal reflux issues [11]. GIT fluid consists of enzymes, acids, and other compounds that quickly decompose drugs, resulting in inadequate absorption and decreased effectiveness. Furthermore, a mucus layer in the GI tract can hinder drug absorption by acting as a barrier, restricting their access to the intestinal epithelium [12, 13]. Thus, using NPs as an oral DDS holds great promise due to their unique properties (which include good therapeutic properties, self-assembly, enhanced biocompatibility, serving as vehicles for antimicrobial agents, controlled drug release, reducing off-target effects, etc.) [2, 4, 14].

Although NPs-based DDS offer promising benefits, their safety must be thoroughly evaluated. The small size of NPs can result in interactions with biological systems that are not yet fully understood, possibly causing toxic consequences. Furthermore, NPs can accumulate in specific tissues or organs, leading to long-lasting damage. A recent study by Cabellos et al. [15] showed that mice exposed to silica NPs suffered inflammation and epithelial damage in the intestines and showed the expression of autophagy proteins [4, 16]. Various mechanisms can contribute to the potential toxicity of NPs. For example, they can cause oxidative stress by generating reactive oxygen species (ROS) that harm cell components [17]. NPs can also trigger inflammatory reactions by activating immune cells, causing tissue injury and malfunction [18]. Moreover, certain NPs might exhibit genotoxic effects by damaging DNA, potentially resulting in mutations and cancer [19]. However, it is crucial to meticulously assess the possible toxicity of NPs to ensure their safety and effectiveness [20, 21]. By refining the development and analysis of NPs, one can reduce toxicity and increase therapeutic advantages by modifying functional groups, coating with cell membranes, or using nanorobots [22]. For example, DDS can be developed using stable intestinal cell-derived exosomes that target colonic cells. It is even known that these exosomes can be found in faces. Lipid NPs derived from plants can also be used as an alternative strategy, thus providing a safe NPs delivery [23, 24]. Thereby, conducting comprehensive pre-clinical and clinical research is vital to evaluate the safety of NPs in the context of orally administered treatments. Such investigations should examine the biodistribution, pharmacokinetics, and toxicity of NPs in both animal models and human subjects [21, 25]. Specifically, close observation of NPs accumulation in different organs and tissues is necessary, along with monitoring any negative impacts on cellular and molecular functions [26]. Therefore, the present chapter delves into the diverse toxicological issues of orally administered NPs (from polymer, lipid, protein, carbon, silica, and metal-based NPs) and summarises the in vitro and in vivo assessments of the toxicity of oral NPs.

2. Structure of GIT and the interaction of nanoparticles

The GIT is a complex system responsible for nutrient absorption, immune function, and waste elimination, divided into several regions, including the mouth, oesophagus, stomach, small intestine, and large intestine. Each region of GIT presents unique anatomical characteristics and physiological conditions that can influence the interaction of NPs. The GIT represents a selective mucosal barrier, with an estimated surface area of 200 m² in adult humans, that can potentially interact with ingested NPs [27]. The anatomical and physiological characteristics of each part of the GIT can affect NPs absorption and elimination [28]. For example, the stomach presents a tough barrier to drug absorption, with a strong acid environment (pH range of 1.0–2.5) that can degrade food, acid-labile drugs, and pathogenic microorganisms. Furthermore, the stomach has extrinsic epithelial cells and a mucin-bicarbonate barrier, which, combined with tight junctions beneath the intrinsic barrier, limits drug absorption. Furthermore, stomach pepsins can inactivate protein drugs [13]. The small intestine has a huge surface area due to the villi and microvilli in the intestinal lumen [29]. The small intestine is considered a major site for oral drug delivery due to its enormous surface and various transport routes [13]. The intestinal mucosa can recognise and transfer ectogenic antigens to the immune system. However, some challenges to drug delivery in the small intestine still arise from its unique physiology [30]. DDS that can increase their retention time in villi and microvilli, improve lipid solubility, and interact with a specific receptor or carrier can increase their overall bioavailability [13, 31]. Therefore, the unique anatomical and physiological features of each part of the GIT play a crucial role in the interaction and potential toxicity of NPs in the GIT.

The interaction of NPs with GIT depends on their physicochemical properties, including size, shape, surface charge, and surface chemistry (Table 1) [58]. Following oral administration, NPs encounter the mucous layer covering the GIT, which protects against harmful agents, such as pathogens and toxins [59]. However, NPs can interact with the mucous layer and penetrate the underlying tissue, resulting in potential toxicity [60].

Nanoparticles composition	Toxic effects	In vitro models	In vivo models	Reference
Polymers such as PLGA, PEG, chitosan, and others	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as HEK293, A549, and others	Mice, rats, and Zebrafish	[32, 33, 34, 35]
Phospholipids, cholesterol, and other lipids	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as HEK293, A549, and others	Mice and rats	[36, 37, 38, 39]
Silicon dioxide or silicon-based compounds	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as HepG2, BEAS-2B, and others	Mice and rats; Caenorhabditis elegans	[40, 41, 42, 43, 44, 45]
Gold	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as MCF-7, HEK293, and others	Mice and rats	[46, 47, 48, 49, 50, 51]
Silver	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as A549, RAW264.7, and others	Mice and rats	[52, 53, 54, 55]
Iron oxide	Cytotoxicity, genotoxicity, oxidative stress, inflammation	Cell lines such as RAW264.7, MCF-7, and others	Mice and rats	[56, 57]

Table 1.

Some studies on the toxic effects of various nanoparticles on different models.

2.1 Influence of NPs size on GIT toxicity

Smaller NPs possess a higher surface area-to-volume ratio, promoting better engagement with the mucous layer and increased absorption into the tissue [61]. Conversely, larger NPs may interact less with the mucous layer and could be eliminated from the GIT before reaching the tissue. Zhao et al. [62] noted that a 100 nm nano-vaccine showed superior pharmacokinetic effectiveness compared to a 500 nm nano-vaccine when an alum adjuvant was administered. The toxicities of NPs are inversely proportional to their size, and they are generally more toxic than large particles of the same chemical substance [63].

2.2 Influence of NPs shape and aspect ratio on GIT toxicity

It was reported that the shape of NPs can impact their association with the GIT as well. NPs with round shapes have been discovered to exhibit a higher level of interaction with the mucous layer compared to alternative forms, such as cylindrical or cubic shapes [61]. This occurs because round NPs possess a larger surface area-to-volume ratio, which can increase their connection with the mucous layer [4]. In addition to size, the configuration and proportion of NPs are essential factors in determining their cytotoxic effects in vivo [64]. Vedhanayagam et al. [65] investigated the impact of various zinc oxide NPs, such as spheres, needle, rod, hexagonal, star, flower, doughnut, circular discs, and cube, on the healing process of wounds. The researchers found that the spherical structure of zinc dioxide (ZnO) within a cross-linked collagen framework leads to improved re-epithelization and more rapid collagen accumulation. A higher aspect ratio of NPs is believed to be linked to increased cytotoxicity due to decreased clearance and enhanced bioavailability of these particles [66]. NPs with higher aspect ratios often exhibit cytotoxicity patterns that resemble those of asbestos. These particles can cause macrophage cell death during phagocytosis and, similar to asbestos fibres, can contribute to cancer formation [67].

2.3 Influence of NPs charge on GIT toxicity

NPs with positive charges have been shown to have a stronger connection with the negatively charged mucous layer compared to those with a negative charge or no charge at all. Researchers have explored using charged polymers to temporarily open tight junctions and enhance drug delivery across the intestinal epithelial barrier. For example, chitosan (a cationic polymer) has been shown to promote the paracellular transport of NPs [68]. NPs’ bioavailability and absorption have been studied only a few times after oral administration with regard to surface charge, hydrophobicity, and shape. NPs with a positive charge demonstrated increased absorption and movement by enterocytes compared to those with a negative or neutral charge, as well as a notable increase in oral bioavailability in vivo [69]. Furthermore, rod-shaped gold NPs (AuNPs) and DNA struggle to permeate or enter cells because of their charge. To enhance uptake, both AuNPs and DNA have undergone surface modification by adding lipid layers, while DNA has also been electrostatically attached to cationic liposomes, facilitating transport into cells [21].

2.4 Influence of NPs surface chemistry on GIT toxicity

Numerous NPs undergo modifications to alter their surface chemistry for specific objectives [70]. For instance, applying a polyethylene glycol (PEG) coating on NPs surfaces to establish water-attracting surface chemistry lessened the intense interaction with mucus components and increased particle movement across the mucus and mucosa [69]. Furthermore, Hasse and colleagues reported that peptide-coated silver NPs (20 nm) were more cytotoxic than citrate-coated silver NPs of the same size. They evaluated cytotoxicity in a human leukaemia cell line using the WST-1 assay [71]. It was perceived that the toxic effects of AgHECp (Silver hexagonal close placed), Ag pristine, and AgPVP (silver polyvinylpyrrolidone) were examined in A431 cell lines (human epidermoid carcinoma) and HaCaT (human keratinocytes) cell lines [71]. It has been revealed that smaller particles with negative surface charge and increased ionic content, such as AgPVP, were highly toxic. Similarly, toxic effects were observed for the larger particles, which were pure AgNP with a negative surface charge and ion content comparable to AgPVP [71]. Additionally, NPs featuring a hydrophobic surface exhibit a more robust interaction with the mucous layer than their hydrophilic counterparts. A cationic polysaccharide with mucoadhesive properties, chitosan has been extensively investigated. Chitosan’s mucoadhesive potency is limited due to its low water solubility and low mucoadhesive strength, so it must be chemically modified to optimise its mucoadhesive properties [72].

3. Toxicological concern with oral nanoparticles

3.1 Polymer-based nanoparticles

Polymer-based NPs (PNPs) can interact with GIT cells and tissues, resulting in inflammation, oxidative stress, and damage to the intestinal barrier. Various poly(lactide-co-glycolic acid) NPs (PLGA) were examined for toxicity in THP-1 macrophages similar to human cells. When used as an NPs stabiliser, chitosan polymer conferred significant cytotoxicity to PLGA NPs, despite being slightly cytotoxic [73]. Research has shown that NPs exhibit varying levels of impact on intestinal cells, intestinal flora, and the intestinal barrier, confirming that NPs cause damage to the digestive system [45, 74]. Polystyrene NPs administration has been found to trigger the TOS-MAPK/NF-kB signalling pathway in macrophage RAW 264.7, leading to inflammation with pro-inflammatory and cytotoxic potential activity [35]. Furthermore, exposure to polystyrene NPs have been shown to disrupt the balance of cell populations within the intestinal cells of zebrafish [75]. Existing data on the toxic effects of ingested zinc oxide (ZnO) NPs on intestinal models need to be more consistent. Several studies have found negative biological effects, such as increased intestinal inflammation, reduced cell viability, and mitochondrial membrane depolarization, resulting from the treatment with ZnO NPs. However, modified ZnO NPs do not cause significant cell damage [76, 77].

3.2 Lipid-based nanoparticles

Lipid-based nanoparticles (LNPs) have gained attention as potential DDS due to their ability to encapsulate hydrophobic medications and shield them from degradation [78]. Cationic lipids, for instance, show significant potential as carriers for delivering delicate substances such as nucleic acids, but certain cationic lipids can lead to cytotoxicity [36]. The impact of hydrophobic chains on lipid toxicity has yet to be thoroughly investigated, impeding the development of less harmful lipids [79]. Solid lipid nanoparticles (SLNs) are among the most prevalent types of LNPs employed in drug delivery. SLNs have been discovered to cause toxicity in the GIT, resulting in inflammation, oxidative stress, and damage to the intestinal barrier [80]. Numerous studies have indicated that the interaction between LNPs and GIT can trigger an immune response, modify the gut microbiota, and induce toxicity [79, 81]. A recent study discovered that oral administration of altered RNA-LNPs led to the expression of pro-inflammatory cytokines (such as interleukin (IL) -6 and macrophage inflammation protein 2 (MIP-2)) and chemokines in the intestines of unexposed mice [39]. Ball et al. [82] documented that LNPs can compromise the stability of the intestinal epithelial barrier and result in intestinal inflammation.

3.3 Silica-based nanoparticles

Silicon-based nanoparticles (SiNPs) can interact with GIT cells and tissues, causing inflammation, oxidative stress, and damage to the intestinal barrier [83]. Various factors influence the toxicity of SiNPs in GIT, including their dimensions, surface area, surface charge, and chemical makeup [84]. Cellular absorption of silica dioxide NPs (SiO2NPs) depends on the size of the particles, particularly in the range of 30-50 nm. SiO₂ NPs within the 30-50 nm size range can be carriers in multiple applications [40, 41]. An in vivo study revealed that ingesting SiO₂NPs led to GIT inflammation and increased permeability, causing intestinal contents to leak into the bloodstream and disrupting the microbiota-gut-brain axis [85]. Guo et al. [83] discovered that exposure to SiO₂NPs affected nutrient transportation, ROS production, barrier function, gene expression, and microvilli structure. To assess the potential toxicity of SiNPs, examining their interactions with gut cells, uptake, and impact on GIT function and microbiota is important. The nanoscale SiO₂ found in the E551 food additive could uniquely impact the absorption and distribution of SiO₂ within the human body [86]. Numerous in vitro studies have shown that SiO₂NPs can produce cytotoxic effects in cultured human cell lines, such as glioblastoma cells, depending on their size, shape, and dose [87]. Furthermore, SiO₂NPs, inhaled or ingested, can infiltrate cells and engage with cellular membranes or organelles, leading to mammalian cell death through oxidative stress, endoplasmic reticulum stress, and apoptosis [42, 43]. A different instance of SiNPs that exhibit toxicity in the GIT involves mesoporous silica NPs (M-SiNPs) [44]. M-SiNPs have been identified to induce toxicity in the GIT, with inflammation, oxidative stress, and harm to the intestinal barrier. SiNPs administration has been found to cause intestinal inflammation by interfering with the hydrolysis and metabolism of nutrient peptides in Caenorhabditis elegans (an invertebrate nematode) [45]. Furthermore, Ogawa et al. [88] revealed that administering 10-nm SiNPs provoked intestinal inflammation by activating an apoptosis-associated speck-like protein with a CARD (caspase activation and recruitment domain) inflammasome.

3.4 Metallic-based nanoparticles

Numerous scientific investigations have been conducted on the therapeutic applications of gold nanoparticles (AuNPs), silver nanoparticles (AgNPs) and superparamagnetic iron oxide nanoparticles (SPIONPs) [55, 89]. They possess several characteristics that make them attractive as DDS. Specifically, they can be easily synthesised in various sizes and shapes, their surfaces can be functionalized with different elements such as polymers, peptides, targeting ligands, imaging probes, and more, and they are generally considered safe for certain in vivo biological applications [55].

AgNPs exhibit distinct properties related to toxicity, surface plasmon resonance, and electrical resistance [90]. In a study by Vandebriel et al. [91], repeated exposure to AgNPs resulted in cytotoxic effects on various rat cells. Intending to create new anticancer treatments, Azizi et al. [92] developed albumin-coated AgNPs, which were found to be specifically taken up by cancerous cells and trigger apoptosis. Furthermore, through numerous pathways, AgNPs are highly effective against gram-positive and gram-negative bacteria. They help address the problem of drug resistance often seen with traditional antibiotics due to their unique mode of action [54]. Clear evidence of systemic toxicity from AgNPs ingested orally or through intravenous routes has been demonstrated, and the toxic effects are related to the amount of Ag⁺ released during exposure [93]. Moreover, the significant surface area of AgNPs that release Ag⁺ ions is a critical aspect that contributes to the cytotoxic behaviour. As widely recognised, smaller AgNPs exhibit a faster pace of silver ion (Ag⁺) dissolution in the nearby microenvironment, owing to their greater surface area-to-volume ratio. This results in increased bioavailability, improved distribution, and increased toxicity compared to larger AgNPs [40, 94]. Research examining the anti-inflammatory properties of AgNPs has shown a considerable decrease in wound inflammation, adjustment of fibrogenic cytokines, a reduction of pro-inflammatory cytokines, and cell death in inflammatory cells [52, 53]. NPs with diameters below 100 nm have been documented to be mainly taken up by endocytosis in epithelial cells. Within these cells, AgNPs can induce oxidative stress, DNA damage, and inflammation [95]. Jeong et al. [96] observed an increase in goblet cells in the intestines and a significant release of mucus granules in mice treated with oral AgNPs (60 nm) at a 30 mg/kg body weight per day for 28 days. Furthermore, AgNPs administered orally (5-20 nm) for 21 days in mice (20 mg/kg body weight) disrupted the microvilli of epithelial cells and affected the intestinal glands [93].

Various in vivo studies have been conducted to assess the possible toxic effects of AuNPs, but the findings still need to be definitive [51]. Factors such as size, shape, surface properties, stabilising coatings, and administration aspects (dosage, duration, and method of administration) can lead to the varying toxic effects of AuNP in vivo [46]. Research has shown that smaller AuNPs (5–15 nm) exhibit a more extensive organ distribution in rodents compared to larger AuNPs (50–100 nm), suggesting a higher risk of toxicity in vivo for smaller AuNPs [47]. Research conducted by Goodman and colleagues [97] indicated that cationic AuNPs interact with the negatively charged cellular membrane, causing damage to the intestinal membrane. Furthermore, citrate-capped AuNP (13 nm in diameter) were harmful to human lung carcinoma cells while not affecting human liver carcinoma cells at the same dose [98]. As additional information is collected, it has been recommended that proper consideration be given to surface chemistry and dosages of Au and AgNPs to utilise them in biomedical applications efficiently [49, 50].

SPIONPs have become increasingly popular in numerous biomedical applications, such as magnetic resonance imaging, targeted drug or gene delivery, and hyperthermia [57]. However, SPIONPs can potentially cause cytotoxicity, negatively impacting vital cellular components such as mitochondria, the nucleus, and DNA [99]. Research examining the influence of various surface coatings on cell behaviour and structure revealed that dextran-magnetite (Fe₃O₄) NPs lead to cell death and decreased proliferation, similar to the effects of uncoated iron oxide particles [56]. Contact with SPIONPs has been linked to considerable harmful consequences, including inflammation, developing apoptotic structures, compromised mitochondrial functioning (MTT), membrane leakage, ROS production, extensive chromosomal aberration, and condensation [57].

4. In vitro tests to evaluate the toxicity of nanoparticles

The in vitro evaluation of the toxicity of NPs serves as a standard approach for identifying acute hazards associated with potentially harmful NPs during this screening process. However, these methods can only partially replace in vivo evaluations. At the same time, in vitro analyses report the immediate harmful effects of NPs in specific cellular settings, in vivo animal models monitor biodistribution and bioaccumulation pathways, which are not accessible through in vitro observations. A comprehensive understanding of NPs toxicity and potential risk requires using both methods [100]. In vitro, NPs toxicity assessment is a critical technique that offers benefits such as reduced cost, quicker results, and minimal ethical issues [101].

4.1 Size and surface charge evaluation

Various analytical approaches exist to examine the toxicological properties of NPs, with two key techniques frequently employed to provide crucial quantitative data: dynamic light scattering (DLS) and zeta potential (ZP) analysis. The ZP and, subsequently, the surface charge of a particle play an important role in inferring potential toxicity, promoting combined DLS-ZP systems as vital tools for preliminary biocompatibility assessments. Other in vitro tests utilised to determine the size and surface charge of NPs encompass scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force spectroscopy (AFM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), surface-enhanced Raman spectroscopy (SERS) and solid-state nuclear magnetic resonance spectroscopy (SSNMR) for compositional analysis, as well as fluorometry for photonic characteristics [102].

4.2 Proliferation assay

Proliferation analysis is conducted by evaluating cell metabolic function to determine cell metabolism using 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the most commonly utilised tetrazolium bromide for evaluating NPs toxicity [103]. This test uses tetrazolium salt to gauge cellular metabolism. The intricate chemical preparation of the MTT assay has led to its substitution for the Alamar blue assay, which is simpler to prepare and measures the cellular redox potential [104]. However, its success has been hindered due to an unclear operating mechanism. Other evaluations include the [3H] thymidine incorporation method to assess cell proliferation, which is typically avoided due to its toxicity [105]. The cologenic assay is another method that counts proliferating cells by visually inspecting them after exposure to NPs [106].

4.3 Apoptosis assay

One primary indicator of nanoparticles (NPs)-induced cell toxicity is apoptosis. This process, triggered by NPs, can lead to cellular renewal and accelerated ageing in mammalian hosts [100]. Commonly used markers for apoptosis include phosphatidylserine (PS), which moves to the cell membrane’s outer layer, and caspase activation. Research has shown that silver NP can induce apoptosis in cells such as mouse embryonic stem cells [107] and stimulate the activation of Caspase 3 and 9 when applied to Drosophila melanogaster larvae [108]. Once host signalling pathways are initiated, active caspases provide various assays, such as cleavable substrates with fluorogenic and chromogenic labels, immunoblotting, immunofluorescence, and affinity assays with connected reporter units [109].

Numerous in vitro tests evaluate NPs toxicity, such as the Annexin V assay, which binds to PS and is a barrier against coagulation cascades. There are also various other techniques for examining apoptosis in cells or tissues exposed to NPs, including the comet assay, the TUNEL (terminal deoxynucleotidyl transferase (TdT) Nick-End labelling) staining technique, and analysing morphological alterations [110, 111, 112]. A decrease in cell size and DNA fragmentation are signs of apoptosis in NPs-treated cells. DNA gel electrophoresis is the most straightforward test for identifying cellular abnormalities. DNA fragmentation with uneven DNA sizes in agarose gel signifies necrosis-mediated cell death, while ladder-like electrophoretic DNA patterns indicate apoptosis-induced cell death in NPs-treated cells [113, 114]. Such changes were observed in human HepG2 hepatoma cells exposed to silica NPs [115]. Furthermore, the single-cell gel electrophoresis assay (SCGE), also known as the comet assay, is used to detect the mutagenic potential of NPs-treated cells by identifying DNA breaks in Drosophila tertiolecta exposed to SiO and TiO NPs [70, 116].

4.4 In vitro assessment of oxidative stress

As a result of the high surface area-to-volume ratio imposed by NPs, enhanced reactivity promotes intracellular damage due to oxidative stress. Detection of oxidative events through ROS measurement, protein carbonyl content, genotoxicity, and inflammatory markers reveal the potential of NPs to produce harmful toxicants in the host [100]. Beyond the natural levels, ROS levels that are produced in the cytoplasm can be detected using ROS-sensitive dyes such as nonionic, non-polar, membrane permeable fluorophore 2′,7′-dichlorofluorescein diacetate (DCFH-DA) [117]. After cellular entry of DCFH-DA, the fluorophore is hydrolysed enzymatically by cytosolic esterase into non-fluorescent polar analogue dichlorofluorescein (DCFH). It is oxidised by cellular ROS into highly fluorescent dichlorofluorescein (DCF) that can be monitored [118]. The use of other agents, such as 2,2,6,6-tetramethylpiperidine (TEMP), detect ROS by reacting with the stable O₂ radical, which can be detected using X-band electron paramagnetic resonance (EPR) [119]. However, the use of DCFH-DA is advantageous over that of TEMP because of its high cost. Other available assays to detect ROS induced due to NPs can be detected using lipid hydroperoxide, Amplex Red assay, measurement of antioxidant depletion by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and superoxide dismutase (SOD) activity by Nitro blue tetrazolium assay [120].

4.5 Necrosis assay

Necrosis disrupts the integrity of the membrane and is commonly used to determine the viability of cells. Necrosis is measured using two dyes Neutral Red and trypan Blue. Neutral red (2-amino-3 methyl-7-dimethyl-amino-phenazonium-chloride) is a weakly cationic supravital dye that, at slightly acid pH, yields a deep red colour [121]. The mechanism of action of neutral red includes diffusion through the plasma membrane and concentrates on the binding of lysosomes through electrostatic, hydrophobic bonds [101]. Any disruption in the cell membrane brought about by NPs results in reduced uptake of neutral red, differentiating live and necrotic cells. Studies have used neutral red to detect necrotic cells induced when exposed to silver NPs [122]. The other dye used for detecting necrotic cells induced through NPs is trypan blue which enters dead cells while being excluded by live cells and thus is used to detect the stability of the cell membrane. Trypan blue has been used to detect the cytotoxicity of cells exerted by Zn NPs [123].

4.6 Viability assays

Lactate dehydrogenase (LDH) is an enzyme produced by living cells that regulates pyruvate and lactate levels through the oxidation of nicotinamide adenine dinucleotide (NAD). When cells are lysed due to the toxic effects of NPs and other agents, LDH is released into the surroundings and maintains the enzyme activity. The usual conversion of pyruvate to lactate in the presence of LDH follows the NADH + pyruvate NAD + + lactate. Spectroscopic methods can monitor the NADH complementary reaction with tetrazolium salts. This time-dependent change in spectroscopic absorbance through enzyme-linked immunosorbent assay (ELISA) tests infers the extent of cellular trauma [100]. Common tetrazolium salts include iodonitrotetrazolium (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) which rival the throughput of the 51Cr assay and exhibit a threefold increase in sensitivity over ultraviolet NADH assays [124]. In addition to these, luminogenic glycylphenylalanyl-aminofluorocoumarin (GF-AFC) and firefly luciferase ATP assays provide non-toxic and highly sensitive options for assessing cell viability [125].

5. In vivo tests to evaluate the toxicity of nanoparticles

In vivo toxicity assessment methods provide more reliable data than in vitro models, and often yield conflicting results [126]. Some examples of in vivo toxicity assessment methods for NPs include biodistribution, clearance, haematology, serum chemistry, and histopathology [101]. For example, researchers have studied the toxicity of aluminium oxide NPs. These NPs have been analysed for their cell toxicity, immunotoxicity, and genotoxicity using in vivo models [126]. Tetrazolium-based assays such as MTT, MTS, and WST-1 have been used to evaluate the cytotoxicity of these NPs [126]. Manganese oxide nanomaterials have also been examined for their potentially harmful effects [127]. One study evaluated the toxicity of a newly synthesised nanomaterial called GNA35 and its precursor Mn₃O₄ using in vitro models representing the respiratory, GIT, and skin systems of the human body [127]. The study provided information on the potential health risks associated with these nanomaterials, highlighting the need for further evaluation using in vivo models [127].

In vivo toxicity assessment of various NPs is crucial for assessing their safety in biomedical and industrial applications. Here are some examples of in vivo toxicity assessment of various NPs:

Polymer NPs, often used for drug delivery systems, have been evaluated for toxicity using in vivo models. A study utilised zebrafish embryos as a model to investigate the toxicity of polystyrene NPs. The study found that NPs demonstrated dose-dependent toxicity, leading to developmental and behavioural abnormalities in zebrafish embryos [46].

Lipid-based NPs are commonly used in drug delivery systems because of their biocompatibility and low toxicity. In a study by Lama et al., researchers evaluated the in vivo toxicity of lipid NPs in rats by measuring their biodistribution, clearance, and histopathology [128]. They found lipid NPs were well tolerated, with no significant adverse effects observed [101].

Protein NPs, such as albumin-based NPs, have been developed for targeted drug delivery. In vivo studies in mice have shown that these NPs can accumulate in tumour tissues and effectively deliver drugs without causing significant systemic toxicity. However, further evaluation of protein NPs using in vivo models is necessary to ensure the safety of these drug delivery systems [129].

Silica NPs have many applications, from drug delivery to cosmetics. In a study by Yu et al., researchers assessed the in vivo toxicity of silica NPs in mice by assessing their biodistribution, clearance, and inflammatory response. They found that silica NPs were distributed primarily in the liver, spleen, and kidneys and induced a transient inflammatory response, suggesting potential risks associated with their use [130].

AuNPs have been studied for their potential applications in various fields, including drug delivery and diagnostics. In vivo toxicity evaluation of gold NPs has been carried out using different animal models. For example, a study investigated the biodistribution and toxicity of AuNPs in mice using various doses and sizes. The results showed that, depending on size and dose, AuNPs could accumulate in organs such as the liver, spleen, lungs, and kidneys, leading to potential toxicity problems [46, 129].

AgNPs have also been investigated for potential applications in medicine and industry. An example of in vivo toxicity assessment of AgNPs is a study that evaluated silver NPs stabilised with gum Arabic protein (AgNPs-GP) in Daphnia, a small freshwater crustacean model. The results indicated that AgNPs-GP exhibited dose-dependent toxicity, causing adverse effects on Daphnia’s survival and reproduction [131].

Iron oxide NPs (SPIONPs) are widely used in biomedical applications, such as drug delivery and magnetic resonance imaging. In a study by Mahmoudi et al., the researchers evaluated the in vivo toxic effects of SPIONPs in mice by observing their biodistribution, retention, and clearance [132]. They found that SPIONPs did not exhibit significant toxicity, but NPs tended to accumulate in the liver and spleen, which can cause long-term adverse effects [57].

6. Conclusion

Throughout the years, the remarkable properties of NPs have led to their recognition as marvels of contemporary science because of their extensive applications. The employment of NPs in the biomedical field has contributed significantly to overcoming numerous diseases and improving the quality of human life. Unlike other delivery methods, orally administering NPs exposes them to various biological and chemical conditions, such as enzyme and microbiota-related digestion and fluctuations in ionic strength and pH levels. Nanotoxicity generally stems from the instability of the NPs and distinct physicochemical properties. Numerous factors, such as the type of NPs, particle and pore size, the extent of modification, frequency and amount of dosage, and administration timing, influence them. Other crucial factors include cell type, cell condition, organ distribution, animal condition, and delivery duration, presenting individual differences in cellular and in vivo contexts. NPs toxicity mechanisms include oxidative stress, inflammation, and apoptosis; however, studies of the implicated signal pathways are still relatively scarce. Due to the numerous variables involved, there currently needs to be a standardised approach to toxicity research. Investigations of NPs toxicity should consider their actual applications and refine safety evaluation indicators for both in vitro and in vivo testing. As observed, a limited amount of data is available for specific NPs types used in DDS. Consequently, before introducing NPs into the therapeutic market, it is essential to evaluate the risk-benefit ratio.