Particuology 69 (2022) 31–48

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Particuology
journal homepage: www.elsevier.com/locate/partic

Invited review

Toxicity of manufactured nanomaterials

Yaping Liu a,b , Shuang Zhu a,c , Zhanjun Gu a,c,∗ , Chunying Chen a,c,d,∗ , Yuliang Zhao a,b,c,d,∗
a
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for
Nanoscience and Technology of China and Institute of High Energy Physics, Beijing 100190, China
b
The First Affiliated Hospital of University of Science and Technology of China, Division of Life Sciences and Medicine, University of Science and Technology
of China, Hefei 230001, China
c
College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China
d
GBA Research Innovation Institute for Nanotechnology, Guangzhou 510700, China

a r t i c l e i n f o a b s t r a c t

Article history: Manufactured nanomaterials with unique properties have been extensively applied in various indus-
Received 25 October 2021 trial, agricultural or medical fields. However, some of the properties have been identified to be closely
Received in revised form related to nanomaterial toxicity. The “nano-paradox” has aroused concerns over the use and develop-
18 November 2021
ment of nanotechnology, which makes it difficult for regulatory agencies to regulate nanomaterials.
Accepted 23 November 2021
The key to fulfilling proper nanomaterial regulation lies in the adequate understanding of the impact of
Available online 29 November 2021
nanomaterial properties on nano-bio interactions. To this end, we start the present work with a brief intro-
duction to nano-bio interactions at different levels. Based on that, how key toxicity-associated properties
Keywords:
Manufactured nanomaterial
of manufactured nanomaterials (i.e., size, shape, chemical composition, surface properties, biocorona
Nanotoxicity formation, agglomeration and/or aggregation state, and biodegradability) impact their toxicokinetics, cel-
Nano-bio interaction lular uptake, trafficking and responses, and toxicity mechanisms is deeply explored. Moreover, advanced
Nanomaterial property analytical methods for studying nano-bio interactions are introduced. Furthermore, the current reg-
Analytical method ulatory and legislative frameworks for nanomaterial-containing products in different regions and/or
Regulation countries are presented. Finally, we propose several challenges facing the nanotoxicology field and their
possible solutions to shed light on the safety evaluation of nanomaterials.
© 2021 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of
Sciences. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction deleterious effects of nanomaterials or nanoproducts on living

organisms throughout their lifecycle, aiming to achieve the reliable
The past decades have witnessed the thriving development and safety evaluation and proper regulation of nanomaterial produc-
extensive applications of nanomaterials or nano-enabled products tion, use, and deposition (Oberdorster et al., 2005). To achieve these
in many fields including energy, aerospace, agriculture, industry, goals, it is a prerequisite to obtaining a comprehensive understand-
and biomedicine, etc. Despite the enormous contributions made by ing of the fundamental toxicological knowledge of nanomaterials.
nanotechnology to human society, there remains a large concern In essence, nanotoxicological studies aim to elucidate how nano-
regarding the nanomaterial toxicity to a wide range of creatures materials interact with organisms at the organ/tissue, cellular and
from environmental species to humans (Ivask et al., 2014; Wu molecular levels, which mainly focus on the toxicokinetics (absorp-
et al., 2019). To better harvest the benefits of nanotechnology tion, distribution, metabolism, excretion (ADME)), cellular uptake
and guardian environmental and human safety, “nanotoxicology” and trafficking, toxicity and underlying mechanisms of nanoma-
as a distinctive field emerged in the early 21st century and has terials. However, there are still large data paucities on nano-bio
been of wide interest since then (Oberdorster, Oberdorster, & interactions at different levels due to the complexity and hetero-
Oberdorster, 2005; Service, 2004). Nanotoxicology deals with the geneity of nanomaterials and biosystems (Wang, Yan, Liu, Chen, &
Zhao, 2018). Fortunately, with the advance in analytical techniques
and research approaches, toxicological results of high quality have
been accumulating (Costa & Fadeel, 2015; Wang et al., 2018).
∗ Corresponding authors at: CAS Key Laboratory for Biomedical Effects of Nano-
In addition to fundamental nanotoxicological research con-
materials and Nanosafety and CAS Center for Excellence in Nanoscience, National
tributed by the scientific communities, the risk assessment and
Center for Nanoscience and Technology of China and Institute of High Energy
Physics, Beijing 100190, China. management of manufactured nanomaterials also ask for inte-
E-mail addresses: [email protected] (Z. Gu), [email protected] (C. Chen), grated endeavors from the industrial communities and regulatory
[email protected] (Y. Zhao).

https://doi.org/10.1016/j.partic.2021.11.007
1674-2001/© 2021 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Liu et al. Particuology 69 (2022) 31–48

bodies for international harmonization and standardization (Fadeel injections, etc. At their portals of entry, various transformations
et al., 2018; Faria et al., 2018). Over the decades, many interna- of nanomaterials such as aggregation/agglomeration, dissolution,
tional and regional organizations have actively launched related formation/evolution of biocorona can take place and affect their
programs or policies to guard the safety of humans and environ- toxicokinetics and biological effects (Cai, Ren et al., 2020; Cai, Liu
ments. In 2006, the Organization for Economic Co-operation and et al., 2020; Misra, Dybowska, Berhanu, Luoma, & Valsami-Jones,
Development (OECD) initiated its Working Party on Manufactured 2012; Setyawati, Zhao, & Ng, 2020). For inhaled, orally uptake
Nanomaterials (WPMN), which “concentrates on the human health and skin topically-applied nanoproducts, the majority are stuck
and environmental safety implications of manufactured nanoma- by complex bioenvironments and reside in the exposure organs,
terials, and aims to ensure that the approach to hazard, exposure where they may further elicit hazardous effects before being grad-
and risk assessment is of a high, science-based, and internation- ually cleared out the body (Cui, Bao, Wang, & Chen, 2020; Larese
ally harmonized standard” via producing OECD test guidelines Filon, Mauro, Adami, Bovenzi, & Crosera, 2015; Osman, Sexton, &
(“Physical-Chemical Decision Framework to inform Decisions for Saleem, 2020). Whereas for the minority of these nanomaterials,
Risk Assessment of Manufactured Nanomaterials”). Similarly, the they can be absorbed into the blood and/or lymphatic circulation
International Organization for Standardization/Technical Commit- (Cui et al., 2020; Larese Filon et al., 2015; Osman et al., 2020). Due
tee 229 (ISO/TC 229) works on standards for nanotechnologies to the slow dose rate, unique absorption routes and special bio-
by establishing several working groups, which include termi- corona acquired/evolved along with their migration from exposure
nology and nomenclature; measurement and characterization; portals to absorption sites, non-intravenously injected nanomate-
health safety and the environment; materials specifications; and rials exhibit more extensive and even distribution across the body.
products and applications (“ISO/TC 229 Nanotechnologies”). As In contrast, their intravenously-injected counterparts are elimi-
suggested by guidelines from these working groups on nanomate- nated from the bloodstream more rapidly and accumulate mostly
rial risk assessment, physicochemical properties of nanomaterials in the mononuclear-phagocytic system (MPS)-rich organs such as
play such a dominant role in eliciting nanotoxicity that they the liver and spleen (Kreyling et al., 2016; Kreyling, Holzwarth,
should be considered throughout the whole toxicological studies Haberl et al., 2017; Kreyling, Holzwarth, Schleh et al., 2017). More-
(“Physical-Chemical Decision Framework to inform Decisions for over, circulating nanomaterials regardless of exposure routes may
Risk Assessment of Manufactured Nanomaterials”). The vital nano- also cross the blood-brain barrier, blood-testis barrier and pla-
material physicochemical properties that are associated with their cental barrier to reach the central nervous system, reproductive
toxic potential include chemical composition, size, shape, surface system and offspring respectively and have biological effects on
chemistry, zeta potential, crystallinity, dimension, solubility, redox these organs (Meng, Leong, Leong, Chen, & Zhao, 2018; Zhang et al.,
potential, etc (Bouwmeester et al., 2011). These properties can indi- 2020). Based on their properties, nanomaterials present various
vidually or synergistically impact different aspects of nanomaterial degradation, metabolism and excretion patterns, which take place
toxicology. Moreover, they may undergo dynamic alterations as mostly in the liver and kidney (Fig. 1(a)) (Wang, He, Zhang, Zhao,
a result of various transformations (e.g., agglomeration, dissolu- & Feng, 2012). According to their toxicokinetic process, it is plausi-
tion, biocorona formation) when the ambient environment changes ble to deduce that a variety of organs especially the lung, intestine,
obviously. For example, the initial size and surface chemistry of liver, spleen and kidney can be the toxicity targets of nanomate-
intravenously-injected nanomaterials change dramatically due to rials. Pulmonary toxicity (inflammation, granulomas, fibrosis), gut
the formation of protein corona, which impacts the subsequent and microbiota dysfunction, liver damage and dysfunction, spleen
blood circulation, biodistribution, metabolism, clearance, cellular inflammation and renal injury, etc. have been validated by numer-
uptake and biological effects (Bai, Wang, Mu, & Su, 2021; Cai & Chen, ous studies (Kermanizadeh, Balharry, Wallin, Loft, & Moller, 2015;
2018; Ding et al., 2018). Therefore, it is significant to thoroughly Mohammadpour, Dobrovolskaia, Cheney, Greish, & Ghandehari,
understand the roles of nanomaterial physicochemical properties 2019).
in mediating different levels of nano-bio interactions, which facil- Following, nano-bio interactions at the cellular, subcellular
itates not only the reliable nanotoxicity assessment but also the and molecular levels provide more fundamental explanations
delicate design of biocompatible nanoproducts. for organ/tissue toxicity and favorable guidance for nanoma-
To this end, the present work focuses on the toxicity of terial safety evaluation (Donahue, Acar, & Wilhelm, 2019).
manufactured nanoparticles to biological bodies in terms of Generally, nanomaterials may adhere to, penetrate across, be
their key physicochemical properties. Firstly, we give a brief embedded in and/or internalized by membranes (Curtis, Bahrami,
overview of how nanomaterials interact with the living organ- Weikl, & Hall, 2015; Lesniak et al., 2013). Cellular internal-
isms at the organ/tissue, cellular and molecular levels from ization of nanomaterials can occur via different pathways,
the toxicological perspective. Based on that, the impact of size, which further impact their intracellular localization, fate and
shape, agglomeration/aggregation state, chemical nature, sur- downstream cytotoxicity (Donahue et al., 2019; Foroozandeh
face chemistry, biocorona formation, and biodegradability on & Aziz, 2018). Endocytosis-based pathways are the predom-
the above-mentioned nano-bio interactions are deeply discussed. inant cellular entry approaches for nanomaterials, including
Moreover, we introduce the major analyzing approaches for study- clathrin-dependent endocytosis, caveolin-dependent endocytosis,
ing the nano-bio interactions at different levels. Furthermore, the clathrin- and caveolin-independent endocytosis, micropinocy-
current regulatory and legislative frameworks of nanomaterial- tosis and phagocytosis (Donahue et al., 2019; Foroozandeh &
containing products in different regions and/or countries are Aziz, 2018). Most endocytosed nanoparticles are firstly entrapped
presented. Finally, the challenges facing the nanotoxicology field by cytomembrane-derived vesicles, which are trafficked to
and possible solutions are proposed to shed light on the safety endosomes and then fuse with lysosomes. In the lysosome, nano-
evaluation of nanomaterials. materials either undergo enzymatic and biochemical degradation,
or achieve lysosomal escape, or be expelled to the extracellu-
Nano-bio interactions at organ, cellular and molecular lar space (Donahue et al., 2019). In contrast, nanomaterials via
levels caveolin-depended endocytosis are confined in caveolae, by which
they transport throughout the cytoplasm and end up in the Golgi
Manufactured nanomaterials in different stages of their lifecy-
apparatus and endoplasmic reticulum (ER). As for nanomateri-
cle and of different usages can be exposed to humans mostly via
als entering cell via direct translocation/diffusion and lipid fusion,
pulmonary inhalation, oral uptake, skin contact and intravenous
or those escaping from the lysosome, they can directly interact

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Fig. 1. Nano-bio interactions at the organ, cellular and molecular levels. (a) Toxicokinetics (i.e., absorption, distribution, metabolism and excretion) of nanomaterials in vivo
(Zhang et al., 2020). Copyright 2020, reprinted with permission from Elsevier. (b) The potential toxicity mechanisms of nanomaterials (Yan et al., 2019). Copyright 2019,
reprinted with permission from John Wiley and Sons.

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Y. Liu et al. Particuology 69 (2022) 31–48

with and disperse across the cytoplasm, thereby gaining possi- weak-embryotoxic but not the nano-ZnO and nano-SiO2 (Farcal
ble access to all organelles including mitochondria and nucleus, et al., 2015). The higher toxicity of nano-ZnO is tightly associ-
etc (Donahue et al., 2019). During intracellular trafficking, nano- ated with its dissolution into toxic Zn2+ , in contrast to insoluble
materials exert cytotoxicity via a variety of toxicity mechanisms, nano-TiO2 and nontoxic degradation products of nano-SiO2 (Farcal
which in essence can be attributed to their oxidative and/or phys- et al., 2015; Ivask et al., 2014; Xia et al., 2008). More recently,
ical damage to cellular components (Fig. 1(b)) (Yan et al., 2019). Mirshafiee et al. compared the toxicological profiling of 29 rare
Specifically, nanomaterials can cause oxidative stress by boost- earth (REOs) and transition metal oxide nanoparticles (TMOs) in
ing the generation of reactive oxygen species (ROS), depleting the different cell types of liver. It was found that pro-oxidative TMOs
intracellular antioxidant systems and/or disturbing the functions elicited apoptosis in hepatocytes, whereas REOs induced pyrop-
of mitochondria (Gajewicz et al., 2015; Nel, Xia, Mädler, & Li, 2006; tosis in Kupffer’s cell and other macrophages. Aside from the
Xia et al., 2008), which results in the oxidative damage of lipid, impact of cell type, the variation in cell death pathway of different
protein and nucleotide acid molecules. Additionally, nanomateri- nanomaterials is presumably ascribed to their different solubil-
als may alter the conformation and function of proteins and DNA, ity, redox and catalytic activity (Mirshafiee et al., 2018). Other
interfere or disrupt the biomembrane systems via direct biophysi- core nanomaterials including nano-CuO, nano-Ag and quantum
cal interaction (Khlebtsov & Dykman, 2011; Ou et al., 2016; Stoehr dots, are capable to dissolve into toxic ions, thus presenting severe
et al., 2011; Wu et al., 2012). Based on the biological function of hazards to biosystems (Sukhanova et al., 2018). In contrast to dis-
damaged cell components, oxidative stress and physical interfer- solvable metal/metal oxide-based nanomaterials, stable metallic
ence can lead to a wide range of downstream deleterious effects, nanomaterials such as Au NPs show more desirable biocompat-
such as cytomembrane leakage, mitochondria dysfunction, lyso- ibility (Farcal et al., 2015; Xia et al., 2008). For instance, it is
some membrane permeabilization (LMP), ER stress, stimulated or reported that Au NPs are not toxic to RAW264.7 macrophages,
blocked signaling pathways involving cell proliferation and death, which reduce the ROS and do not induce the secretion of proin-
cytoskeleton disruption, genotoxicity, etc., which may finally result flammatory cytokines (Shukla et al., 2005). In contrast to the fact
in inflammatory response, cell cycle arrest, and cell death via dif- that many soluble metallic nanomaterials exert relatively high
ferent ways including apoptosis, necrosis, autophagy, ferroptosis toxicity via releasing poisonous metal ions and catalyzing ROS gen-
and pyroptosis (Magdolenova et al., 2014; Ou et al., 2016; Stern, eration, most nonmetallic nanomaterials possess lower toxicity
Adiseshaiah, & Crist, 2012; Wang & Chen, 2016; Wu et al., 2012; and distinct underlying mechanisms. The low toxicity of meso-
Yan et al., 2019; Xu, Zheng et al., 2020; Xu, Lu et al., 2020; Yan et al., porous silica nanoparticles (MSNs) is partly derived from their
2019). unique surface silanol groups, which can either form hydrogen
bonds or dissociate into SiO− to electrostatically interact with vari-
ous biomolecules, thus triggering the disruption of cytomembrane,
Key toxicity-associated properties of nanomaterials
lysosome and mitochondria, etc (Tarn et al., 2013). Another rep-
resentative nanomaterial with good biocompatibility is liposomes,
Nano-bio interactions at different levels are influenced by
which have been translated into the clinic as drug carriers (e.g.,
several key properties of nanomaterials including the chemical
Doxil® and AmBisome® ) to alleviate the toxicity and improve the
nature of core nanomaterials, impurities, functionalization strate-
pharmacokinetics of active pharmaceutical ingredients (Beltrán-
gies, surface properties (surface charge/hydrophobicity/ligands),
Gracia, López-Camacho, Higuera-Ciapara, Velázquez-Fernández, &
size, shape, agglomeration, and biocorona, etc. To deeply under-
Vallejo-Cardona, 2019). In addition, a library of nanomaterials with
stand nanotoxicity, in this section, we dedicate to illustrating the
unique enzyme-mimicking capability termed as “nanozyme”, has
toxicological impacts of various nanomaterial properties, especially
gained intense interest from the biomedical, energy, food safety and
how these properties affect the toxicokinetics, cellular uptake, and
environment science fields. According to their enzyme-mimicking
toxicity mechanisms of nanomaterials. The solid examples used in
ability, nanozymes can be divided into anti-oxidant nanozymes
this section are mostly chosen from 11 types of nanomaterials pro-
and pro-oxidant nanozymes, both of which have shown relatively
posed by WPMN that are either currently in use or will be in use
good biocompatibility and even protective effects (Dai, Ding, & Li,
soon, including cerium oxide (CeO2 ), zinc oxide (ZnO), titanium
2021; Singh, 2019). For example, CeO2 NPs, a representative of anti-
dioxide (TiO2 ), gold nanoparticles (Au NPs), silver nanoparticles
oxidant nanozymes with superoxide oxidase (SOD) and/or catalase
(Ag NPs), fullerenes, multi-walled carbon nanotubes (MWCNTs),
mimetic activity can scavenge superoxide and/or hydrogen perox-
single-walled carbon nanotubes (SWCNTs), nanoclays, silicon diox-
ide (H2 O2 ) under different conditions, which make them potential
ide (SiO2 ) and dendrimers (“Testing Programme of Manufactured
therapeutics for anti-inflammation, antioxidant, pro-proliferation
Nanomaterials – Overview”). Apart from that, other widely used
and neuroprotection with limited toxicity (Wei & Wang, 2013).
manufactured nanomaterials such as liposome, graphene family,
Although pro-oxidant nanozymes such as iron-based nanozymes,
MoS2 nanosheet, etc. are also employed as examples when illus-
can display peroxidase mimetic activities to generate destructive
trating property-associated nanotoxicity.
hydroxyl radicals, their substrates of (i.e., H2 O2 ) and catalytic con-
ditions (i.e., acidic pH) often exist in disease conditions (e.g., tumor
Chemical composition cells), thus endowing them selectively killing capability towards
tumor cells instead of healthy tissues (Li, Zhang et al., 2020; Li,
The chemical nature of nanomaterial core is a vital factor for Wang et al., 2020).
nanomaterial toxicity, which determines their dissolution, redox Besides the chemical nature of core nanomaterials, intentional
capability, ionization properties, affinity to macromolecules, and material design such as surface coating/grafting and element dop-
thus their toxicity potential and underlying mechanisms. For exam- ing would impact the toxicology of nanomaterials. The impact of
ple, a comprehensive in vitro toxicity testing of representative surface modification will be discussed in the next section, here
nanomaterials was conducted in 12 cellular models representing we focus on how element doping impacts the safety of nanoma-
different organs/systems via 10 different assays for cytotoxicity, terials. Element doping refers to the functionalization process in
embryotoxicity, epithelial integrity, cytokine secretion and oxida- which a small percentage of foreign atoms, mostly metal atoms
tive stress (Farcal et al., 2015). The hazard ranking has been are introduced into pure nanomaterials. Doping could modulate
established as nano-ZnO > nano-SiO2 > nano-TiO2 in most tissues, the electronical, magnetic, optical and redox properties of nano-
except for the embryotoxic tissue, where nano-TiO2 was found materials via altering their Fermin energy level and bandgap (Yan

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Y. Liu et al. Particuology 69 (2022) 31–48

et al., 2019). Generally, doped nanomaterials possess enhanced nor permeate the skin (Larese Filon et al., 2015). Since the MPS
capability for generating ROS owing to the improved exposure of system exhibit different phagocytic capabilities towards nanoma-
catalytically active surface atoms. Doping elements with higher terials of different sizes and the microstructures of various organs
migration likelihood to nanomaterial surface and stronger catalytic such as the live, spleen and kidney set up size restrictions for
activity exhibit higher catalytic activity, thus rising higher extent nanomaterials, their blood circulation, biodistribution and elimina-
of oxidative stress (Le et al., 2016). Furthermore, rational doping tion profiles are greatly impacted by their hydrodynamic size after
strategies can form heterojunction structures between the doping entering the bloodstream (Zhang et al., 2020). In general, nano-
materials and pristine nanomaterials, which facilitate the electron- materials smaller than 6–8 nm can be efficiently filtered by the
hole separation and electron transfer kinetics, thereby elevating cut-off glomerular podocyte silt, thus exhibiting short circulation
their ROS generation capability (Yan et al., 2019; Zhang et al., 2014). duration and rapid renal clearance (Wang & Liu, 2018). However,
Moreover, the complicated implications of doping on nanotoxicity ultrasmall nanomaterials with sizes in the sub-nanometre regime
are related to their modulation on nanomaterial dissolution. For (e.g., Au nanoclusters less than 1 nm) exhibit dramatically reduced
instance, iron doping can alleviate the in vitro and in vivo toxicity of renal clearance efficiency due to their easier entrapment by the
ZnO nanoparticles such as loss of mitochondrial membrane poten- gel filtration chromatography-like glomerular glycocalyx (Du et al.,
tial and membrane disruption, interference in zebrafish embryo 2017). In contrast, nanoparticles larger than 200 nm are not able
hatching, inflammatory and oxidative damage to rodent lung by to extravasate through the discontinuous hepatic/splenic sinu-
slowing the dissolution of nano-ZnO (George et al., 2010; Xia et al., soidal epithelium. Together with the slow blood flow and abundant
2011). macrophages in liver and spleen sinusoids, these nanoparticles can
The safety profiles of nanomaterials are also impacted by other be rapidly removed from the blood and mainly accumulated in the
substances contained in nanomaterials such as impurities. It has liver and spleen (Wang et al., 2012). Besides, 10–20 nm nanomate-
been reported that metal catalyst residues (e.g., Fe, Y, Ni, Mo, etc.) rials undergo rapid hepatocyte uptake followed by bile excretion
introduced during the fabrication and/or acid treatment of CNTs are (Wang et al., 2012). Therefore, nanomaterials of the rest size range
one of the most important sources of CNT toxicity. These metal ions (i.e., 20−200 nm) are most likely to not only avoid the rapid renal
can boost ROS generation and interfere with intracellular antiox- or hepatobiliary clearance, but also escape the phagocytosis of MPS
idant systems, thus leading to or aggravating cellular oxidative systems, thus presenting relatively extended blood circulation and
stress and damage (Liu, Zhao, Sun, & Chen, 2013). Likewise, the whole-body distribution (Wang et al., 2012).
toxicity of cetyltrimethylammonium bromide (CTAB)-coated Au At the cellular level, numerous experimental, theoretical and
nanomaterials is largely attributed to the detached or residual free computational studies showed that the dominant pathway of
CTAB (Carnovale, Bryant, Shukla, & Bansal, 2019). The so-called nanomaterial cellular entry is size-dependent (Donahue et al.,
nanotoxicity may also result from unexpected organic contamina- 2019; Foroozandeh & Aziz, 2018; Lunnoo, Assawakhajornsak, &
tion. For instance, Chen. et al. prepared MoS2 nanosheets via direct Puangmali, 2019). Small nanoparticles are believed to directly cross
sulfurization of molybdenum thin film on a quartz plate, which the cell membrane and translocate to the cytoplasm, however,
avoids potential organic contamination and justifies the low toxic- the explicit small size effect is still under exploration. For large
ity of uncontaminated MoS2 nanosheets (Chen et al., 2018). One of nanomaterials, various endocytic pathways are the major cellular
the most common and toxicity-related contaminants is endotoxins uptake routes (Lunnoo et al., 2019). After nanomaterial entrapment,
or lipopolysaccharide (LPS), which is resistant to most sterilization caveolin- and clathrin-dependent endocytosis generate 50–100 nm
methods in laboratories and can bind to a wide range of materi- caveolae and 100–500 nm intracellular vesicles respectively, which
als including nanomaterials and laboratory equipment. The large are the main pathways for nanomaterials of tens of or a few
amounts of endotoxins contained in nanomaterials can activate hundred nanometers (Donahue et al., 2019; Foroozandeh & Aziz,
Toll-like receptors 4 (TLR4) of various immune cells, thus result- 2018). In contrast, macropinocytosis forms leaky macropinosomes
ing in enhanced proinflammatory cytokine release. It is likely to (0.5–1.5 ␮m) to facilitate the transportation of large nanomaterials
make misinterpretations of the inflammation-inducing capability (250 hundreds of nanometers) (Donahue et al., 2019; Foroozandeh
of pristine nanomaterials, considering their contamination by LPS & Aziz, 2018). Notably, size itself is not the only determinant
(Himly et al., 2020). for nanomaterials’ cellular uptake, other factors such as surface
hydrophobicity, ligands, cell type, etc. also play important roles
Size in this scenario. For example, it has been reported that 40−50 nm
Herceptin-modified gold nanoparticles (Her-GNPs) had a much
Size is another vital factor of nanomaterials that significantly higher extent of cellular uptake than that of Her-GNPs with smaller
impacts the whole induction process of nanotoxicity (Zhang et al., (2–10 nm) and larger (80–100 nm) size. This was explained that
2020). Various nanomaterials display size-dependent absorption tiny Her-GNPs possess low ligand density, weak binding avidity
via different exposure pathways. In general, orally administrated with receptors and fast diffusion rate, leading to easy disassociation
nanomaterials can be absorbed into the circulation system via the with receptors before cell engulfment, whereas although large Her-
transcytosis of enterocytes (for nanomaterials less than 50 nm), GNPs form multiple binding with receptors, they are too large for
paracellular transportation through the tight junction between the membrane wrapping (Fig. 2) (Jiang, Kim, Rutka, & Chan, 2008).
enterocytes (for nanomaterials of 10 nm or less), and phagocytosis Furthermore, nanomaterial size is tightly related to their capa-
by M-cells lining Peyer’s patches (for nanomaterials of 50–500 nm) bility for cytomembrane attachment, penetration and disruption,
(Powell, Faria, Thomas-McKay, & Pele, 2010; Setyawati et al., 2020). thereby impacting the membrane integrity and functions (Behzadi
Inhaled nanomaterials with less than 100 nm in size can eas- et al., 2017; Farnoud & Nazemidashtarjandi, 2019). For instance,
ily deposit in the alveolar-capillary bed, where nanomaterials or large graphene oxide (GO) was reported to strongly adsorb onto
agglomerates larger than 70–100 nm are mainly engulfed by alveo- the surface of macrophage cytomembrane, which could trigger less
lar macrophages, and smaller nanomaterials can penetrate through phagocytosis but more robust interaction with TLRs and activation
the air-blood barrier via paracellular and/or transcellular transport of pro-inflammatory pathways. In contrast, small GO are mostly
(Oberdorster et al., 2005; Osman et al., 2020). Similarly, skin top- phagocytized by macrophages, which may induce oxidative stress
ically applied nanomaterials <4 nm can penetrate and permeate inside macrophages (Ma et al., 2015). What’s more, the ability of
intact skin, those of 21–45 nm can penetrate and permeate only nanomaterial to cause oxidative stress, genotoxicity, mitochondrial
damaged skin, whereas those with size >45 nm cannot penetrate and lysosomal dysfunction, cell cycle arrest and cell death, etc. is

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Y. Liu et al. Particuology 69 (2022) 31–48

Fig. 2. Size has complex effects on the cellular uptake of Herceptin-modified gold nanoparticles (Her-GNPs). (a) Illustrations with corresponding fluorescence images of ErbB2
receptor localization after treatment with different-sized Her-GNPs. Arrows indicate ErbB2 receptors, and the nucleus is counterstained with DAPI (blue) (scale bars = 10 ␮m).
(b) Cross-sectional fluorescence intensity measurements of ErbB2 receptor localization patterns with G2 and G40 Her-GNPs (scale bars = 10 ␮m). (c) Surface ErbB2 expression
analysis using untreated cells normalized as 100% expression level. Cells were treated with unmodified 40-nm GNPs, Herceptin and Herceptin-modified GNPs of various
sizes (* denotes statistical significance for G40/G50 compared to Her-GNPs of other sizes, p < 0.05, ANOVA) (Jiang et al., 2008). Copyright 2008, reprinted with permission
from Springer Nature (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

also found to be size-dependent (Ahmed et al., 2017; Pan et al., excretion, irrespective of intravenous injection or oral administra-
2007). Generally, smaller nanomaterials show higher toxic poten- tion (Huang et al., 2011; Li, Liu et al., 2015; Li, Kroger et al., 2015; Li,
tial than their larger counterpart, which can be attributed to their Zhao et al., 2015). As for in vivo toxicity after oral ingestion, all MSNs
enhanced surface area, exposed surface atom ratio and thus ele- were found to exert no obvious organ damages except for renal tox-
vated catalytic capability (Nel et al., 2006). Besides, shrinkage in icity such as renal tubular necrosis and hemorrhage, with kidney
size may damage structured crystal plane and electronic configu- damage becoming more severe as the aspect ratio decreases (Li, Liu
ration to give rise to more reactive surface sites, leading to higher et al., 2015; Li, Kroger et al., 2015; Li, Zhao et al., 2015). In addition
ROS generation capability and hence hazardous effects (Nel et al., to impacting the in vivo biofate and organ toxicity, shape is a key
2006). factor for their cellular uptake and cytotoxicity. A computational
simulation reported that under the conditions of the equal surface
area of the cores, the same amount of grafted PEG polymers and
Shape
identical ligand-receptor binding strength, nanospheres presented
the fastest endocytosis rate, followed by nanocubes, nanorods and
Nanomaterials can be fabricated in a variety of shapes, cate-
nanodisks. It was explained that symmetrical nanosphere requires
gorized as nanosphere, nanotube, nanorod, nanowire, nanocube,
the minimal membrane binding energy change, whereas nanorods
nanosheet, etc (Demir, 2021; Sukhanova et al., 2018). The shape can
and nanodisks undergo complex rotation depending on their entry
influence the toxicokinetics and in vivo toxicity of nanomaterials
angle, and cause a large extent of membrane deformation, thus
(Huang et al., 2011; Li, Liu et al., 2015; Li, Kroger, & Liu, 2015; Li, Zhao
slowing the endocytic speed (Li, Liu et al., 2015; Li, Kroger et al.,
et al., 2015). For instance, it is reported that an increase in the aspect
2015; Li, Zhao et al., 2015). Another experimental research indi-
ratio of rod-like MSNs can decrease their liver distribution and renal

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Y. Liu et al. Particuology 69 (2022) 31–48

cated that spherical CTAB-stabilized gold nanomaterials (AuNS) was mostly attributed to their stronger electrostatic interac-
exhibited the lowest cell viability, followed by prismatic shaped tion with negatively-charged cytomembrane and the formation
nanogold (AuPr), in contrast to the comparable good bioavailability of nanopores in the cytomembrane (Foroozandeh & Aziz, 2018;
of the cubic and rod-shaped nanogold in the concentration range Zheng, Mortensen, Ravichandran, Bentley, & DeLouise, 2017). How-
of 10–100 ␮M (Carnovale et al., 2019). The extremely high cyto- ever, the simple electrostatic interaction theory only accounts
toxicity of AuNS was partly attributed to their significant levels for part of the implications of surface properties to cellular
of cellular uptake. However, despite the lowest level of cell inter- uptake. Notably, surface properties can indirectly decrease or
nalization, AuNPr also caused high levels of cell death, which may promote the cell internalization of nanomaterials mediated by
derive from their sharp edges-caused cellular structure damage their absorbed biomolecules. For example, Cai et al. reported that
(Carnovale et al., 2019). In addition, the different shapes may lead to biocorona mediated by CTAB, poly(diallyl dimethyl) ammonium
different exposure of crystal plane, biocorona formation, catalytic chloride (PDDAC), and PEI modification had a positive influ-
capability, thus exhibiting distinct toxic potential (Carnovale et al., ence on the macrophage phagocytosis of gold nanorods (NRs),
2019; Mostafa et al., 2010). whereas polystyrene sulfonate (PSS)- and PEG-mediated bio-
corona decreased the endocytosis of gold NRs (Cai, Ren et al.,
Surface properties 2020; Cai, Liu et al., 2020). Moreover, different surface proper-
ties can lead to different intracellular trafficking and localization.
Since nano-bio interactions often take place at the interface Modifying nanomaterials with surface ligands (e.g., folic acid, albu-
between nanomaterials and their ambient bioenvironment, surface min and cholesterol) can trigger caveolin-mediated endocytosis
properties have significant impacts on their behaviors and bio- (Blanco, Shen, & Ferrari, 2015), followed by the transportation
fate in the environment and organisms (Kim, Saha, Kim, & Rotello, of nanomaterials through the cytoplasm, achievement of lysoso-
2013). These key toxicology-related surface properties include sur- mal escape and termination in ER and Golgi apparatus (Donahue
face charge, surface hydrophobicity and surface atoms/groups, etc. et al., 2019). Nanomaterials with excessive positive charges such
Notably, in the biomedical field, in order to improve their col- as PEI dendrimers can dramatically change the osmolarity of var-
loid stability, biocompatibility/safety and pharmacokinetic profiles, ious cytosolic vesicles, which may induce swollen and ruptured
the pristine nanoparticles would undergo surface modifications endosomes/lysosomes, thus being released into the cytoplasm and
after synthesis, which may alter the primary interfacial prop- getting access to all kinds of organelles and even the nucleus
erties of core nanoparticles and thus their toxicological profiles (Benjaminsen, Mattebjerg, Henriksen, Moghimi, & Andresen, 2013).
(Kim et al., 2013; Yan et al., 2019). Endowing nanomaterials with Besides, surface ligands including glutathione (GSH) and triphenoyl
hydrophilic and electro-neutral surfaces can facilitate their mucus phosphonium endow nanomaterials with mitochondrial targeting
permeating ability, by avoiding their interactions with hydropho- capability, whereas modifying nanomaterials with triamcinolone
bic and negatively-charged mucin network of mucosal barriers acetonide and nuclear localization sequence peptide facilitates
in the gastrointestinal tract (GIT) and other mucosas. In contrast, their access to the nucleus (Donahue et al., 2019; Zheng et al.,
the penetration across epithelium requires nanomaterials with 2017). Furthermore, surface properties have direct effects on the
hydrophobic and positively-charged surfaces. Therefore, surface induction of cytotoxicity. Manshian et al. found that higher levels
properties have complex impacts on the absorption of nanomate- of surface hydrophobicity correlate positively with the increased
rials (Wu, Jiang, & Zhang, 2018; Wu, Shan, Zhang, & Huang, 2018). cell membrane damages and elevated induction of autophagy
Moreover, surface properties also exert effects on the biodistri- (Manshian et al., 2014). Liu et al. reported that different sur-
bution and elimination of nanomaterials, which is deeply rooted face charges and ligands would exert different interference in
in their significant influence on biocorona formation, agglomer- cell metabolic processes. In contrast to non-obvious cytotoxic-
ation state and thus their colloid/disperse stability in vivo (Li, ity of PDDAC-Au NRs, positively-charged PEI-Au NRs exhibited
Zhang et al., 2020; Li, Wang et al., 2020; Visalakshan, Cavallaro severe cytotoxicity to A549 cells due to their widespread dis-
et al., 2019; Visalakshan, MacGregor et al., 2019). For instance, ruption to energy metabolism, choline metabolism, hexosamine
polyethylene glycol (PEG) modification makes bare gold nanopar- biosynthesis and oxidative stress pathways (Liu, Wang, Zhang,
ticles well-dispersed individual particles in the blood and endows Wang et al., 2016; Liu, Wang, Zhang, Wu et al., 2016). Xu et al.
them with relative resistance to opsonization, which results in discovered that the higher density of surface vacancies in WS2
a more even distribution of PEG-GNPs across the body with less and MoS2 nanosheets results in the greater amount of active W
extend accumulation in the liver and spleen than that of polyethy- and Mo atoms and therefore plays a decisive role in their capa-
lene imide (PEI)-GNPs. Moreover, well-dispersed PEG-GNPs with bility of inducing ferroptosis. After healing the surface vacanies
small hydrodynamic sizes are capable of extravasating through the in WS2 and MoS2 nanosheets by Na2 S and methanol treat-
discontinuous liver/spleen sinusoidal endothelium (100–200 nm) ment respectively, surface passivated nanosheets exhibited the
and even glomerular endothelium, thus contributing to their larger reduced destructive interaction with lysosomes and thus alli-
extent of hepatobiliary and renal excretion. In contrast, unstable vating ferroptotic cell death (Xu, Zheng et al., 2020; Xu, Lu
PEI-GNPs may undergo PEI detachment and/or protein absorption, et al., 2020). Moreover, the ROS generation capability and release
which facilitates their agglomeration and the strong sequestration extent of toxic metal ions from the core nanomaterials are also
by MPS cells (e.g., Kuppfer cells) in vivo. Therefore, PEI-GNPs are affected by the surface properties of nanomaterials. Quantum
mainly accumulated in MPS-rich organs (i.e., the liver and spleen) dots (QDs) with different surface coatings exhibit different ROS
with little excretion via hepatobiliary or renal routes, which result generation capabilities, ranked as follows: PEI-QDs > dihydrolipoic
in severer liver damage and interference with metabolic process acid (DHLA)-QDs > GSH-QDs ≈ no treatment ≈ cysteamine (CYS)-
and immune response (Fig. 3) (Li, Zhang et al., 2020; Li, Wang et al., QDs (Zheng et al., 2017). PEG- and bovine serum albumin
2020). (BSA)-coated Ag NPs were reported to reduce ROS generation and
In addition to impacting the toxicokinetics of nanomaterials, protect intracellular antioxidant systems, thus presenting lower
surface properties are key determinators for cellular uptake, intra- toxicity than bare Ag NPs at both in vitro and in vivo levels
cellular trafficking and the consequence cytotoxicity. Numerous (Das et al., 2017). The alleviated oxidative stress and cell death is
in vitro studies have proved that positively-charged nanoma- attributed to the strong inhibitory effects of PEG/BSA coatings on
terials exhibit a higher extent of cellular internalization than Ag ions dissolution and leaching into bioenvironments (Das et al.,
that of negatively-charged or neutral nanomaterials, which 2017).

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Y. Liu et al. Particuology 69 (2022) 31–48

Fig. 3. Surface coatings impact the dispersibility, organ distribution and excretion pathways of PEG-GNPs and PEI-GNPs. TEM imaging of PEG-GNPs (a1 ) and PEI-GNPs (b1 ).
PEG-GNPs were distributed over all organs of the body with the liver, spleen, kidney and small intestine as the major accumulating sites (a2 ); PEI-GNPs were sequestered
mainly by the liver, spleen, and lung (b2 ). A small number of PEG-GNPs were engulfed by Kuppfer cells (a3 ), whereas a large number of clustered PEG-GNPs were deposited in
the Disse space (a4 ); a large number of PEI-GNPs were deposited in the cytosol of Kuppfer cells (b3 ) while little was found in Disse space (b4 ) after 1 h intravenous injection. A
small number of PEG-GNPs were found deposited in the tubulointerstitium (a5 ) and were taken up into the lysosomes of renal tubule epithelial cells (a6 ), whereas PEI-GNPs
accumulated in podocytes (b5 ), few of which crossed the glomerular basement membrane and were trapped in the lysosomes of renal tubule epithelial cells (b6 ) after 1 h
intravenous injection (Li, Zhang et al., 2020; Li, Wang et al., 2020). Copyright 2020, Li et al., open access.

Toxicity associated-biotransformation of nanomaterials in the MPS-rich organs (e.g., the liver and spleen), and can not
be efficiently eliminated via either hepatobiliary or renal path-
Agglomeration or aggregation state ways (Li, Zhang et al., 2020; Li, Wang et al., 2020). In contrast,
Due to their ultrahigh surface energy, nanomaterials are individual nanoparticles exhibit more extensive distribution across
extremely unstable in the environment and biocontexts, which the whole body and faster excretion (Li, Zhang et al., 2020; Li,
tend to undergo a series of transformations to lower their sur- Wang et al., 2020). Moreover, once the apparent size of particles
face energy. Forming agglomerates and/or aggregates is one of exceeds 250 nm, the likelihood for their tissue transportation from
the most common transformations (Cai, Ren et al., 2020; Cai, Liu the bloodstream is very low to negligible due to the obstruction of
et al., 2020). Agglomeration and aggregation both refer to the pro- various blood vessels and organs (Bruinink et al., 2015). At the cellu-
cess in which primary nanoparticles gather together to generate lar level, agglomeration state can alter the size-dependent cellular
a group of secondary particles. However, agglomerates rely on internalizing pathways and uptake degree of primary nanomate-
weak interaction forces (e.g, van der Waals forces and electrostatic rials (Bruinink et al., 2015; Lunnoo et al., 2019). In most cases,
forces) and can disintegrate in bioenvironments, whereas aggre- agglomerates display reduced cell uptake compared with pris-
gates are the results of long-time agglomeration, leading to fused tine nanomaterials due to size enlargement (Bruinink et al., 2015).
aggregates by strong forces such as covalent or metallic bonds However, for specific cells especially macrophages, the increased
(Spurgeon, Lahive, & Schultz, 2020; Zook, MacCuspie, Locascio, size of agglomerates can facilitate the cellular delivery of small
Halter, & Elliott, 2011). Agglomeration and/or aggregation can take pristine nanomaterials, thus eliciting severer toxicity (Murugadoss
place before and after human exposure. This state is highly dynamic et al., 2020). Similarly, the impact of agglomeration state on the
and unstable, which is affected by pH, ion strength, cholate, and ultimate nanotoxicity still remains controversial. In most cases,
proteins, etc (Bruinink, Wang, & Wick, 2015; Spurgeon et al., forming agglomerates/aggregates is believed to alleviate the haz-
2020). In essence, the impact of agglomeration and/or aggrega- ardous potential of pristine nanomaterials, which is owing to their
tion on the toxicokinetics, cellular uptake, toxicity of nanomaterials decrease in specific surface area and thus nano-cell interactions
can be attributed to their enhancement in nanomaterial appar- (Allegri et al., 2016; Bruinink et al., 2015; Sager et al., 2016). The
ent size. Agglomeration formed in the environment is believed reduced active surface atoms may result in lower ROS generation,
to greatly reduce human exposure and subsequent translocation slower dissolution of soluble nanomaterials and decreased release
from primary barriers such as lung, GIT and skin due to their of toxic ions (Zook et al., 2011). However, there are also studies
size limitation, thus protecting humans against the potential sys- reporting elevated toxicity after agglomeration. For example, rope-
temic toxicity (Bruinink et al., 2015). However, once entered into like agglomerated CNTs exhibited a higher extent of cytotoxicity
or formed within the biosystems, agglomerates/aggregates seem than asbestos fibers and well-dispersed CNT-bundles at the same
to have high toxic potentials. Subchronic 13-week inhalation of concentrations, presumably because CNT agglomerates were big-
MWCNTs caused pathological changes in both upper and lower res- ger, stiffer and more solid, thus eliciting more pronounced damage
piratory tracts at overload conditions, which were attributed to the to the cell structures (Wick et al., 2007).
high density of CNT agglomerates (Pauluhn, 2010). Agglomeration
state can also impact the biodistribution and elimination patterns Biocorona formation/evolution
of nanomaterials. Unstable intravenously injected nanomaterials Another transformation of primary nanomaterials to lower their
tend to form large agglomerates, which are mostly sequestered ultrahigh surface energy is by quickly absorbing a selected group of

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Y. Liu et al. Particuology 69 (2022) 31–48

biomolecules onto their surfaces, termed biocorona (Cai, Ren et al., alleviating toxic surfactant-induced cytotoxicity and prohibiting
2020; Cai, Liu et al., 2020; Monopoli, Åberg, Salvati, & Dawson, nanomaterials dissolution and the subsequent release of toxic
2012). The formation of biocorona is highly dependent on the metal ions, etc (Cai & Chen, 2018; Choi, Riviere, & Monteiro-Riviere,
physicochemical properties of primary nanomaterials (e.g., size, 2017; Liu et al., 2020). Nevertheless, nanomaterials can maintain
surface chemistry, shape, etc.) and their ambient biocontexts (Cai or alter the conformation of the attached proteins, thus mod-
& Chen, 2018; Monopoli et al., 2012). The absorbed biocorona ulating immune responses (Visalakshan, Cavallaro et al., 2019;
changes the synthetic identity of nanomaterials and endow them Visalakshan, MacGregor et al., 2019). Planar MoS2 nanosheets do
with a brand-new biological identity, which plays a predominant not alter the conformation of the absorbed IgG in protein corona,
role in mediating various nano-bio interactions, including absorp- which interacts with Fc receptors on macrophages, thus activat-
tion, blood circulation, distribution, metabolism, cellular uptake ing the NF-␬B signaling pathway and proinflammatory cytokine
and toxicity (Bai et al., 2021; Baimanov et al., 2020; Cai & Chen, release (Baimanov et al., 2020). Nanotopography with 68 nm
2018; Ding et al., 2018; Stepien et al., 2018; Wang et al., 2019). nanogold immobilized to a polymer film induced the attached
Notably, biocorona with relatively stable and specific inner hard fibrinogen to unfold, which activated Mac-1 positive monocytes
layer and loosely-bounded and highly-dynamic outer soft layer, can and thus inflammatory response, whereas nanotopography of
evolve along with their transportation across the body (Monopoli 38 nm nanogold reduced these responses (Visalakshan, Cavallaro
et al., 2012), thus making their biological impacts highly compli- et al., 2019; Visalakshan, MacGregor et al., 2019). Aside from their
cated. immunomodulation effects, biocorona on nanomaterials can alter
Biocorona exerts profound influences on nanomaterial bioki- the gene expression involving phase I metabolism and phospho-
netics and biofate (Stepien et al., 2018). As a general rule, the lipidosis in human hepatocytes (Choi et al., 2017), robustly activate
rich absorption of desoponins such as albumin and apolipoprotein the ER stress pathways in rat aortic endothelial cells (Persaud et al.,
(Apo) increases the resistance of nanomaterials to the sequestra- 2019), or trigger cell apoptosis via activating AKT/caspase 3 path-
tion of MPS cells, which leads to their long blood circulation and way (Wu, Jiang et al., 2018; Wu, Shan et al., 2018), induce the
even accumulation in the whole body. In contrast, absorbing a development of fibrosis in mice lung via prolonged activation of
large number of opsonins (e.g., immunoglobulins, fibrinogens and TGF-␤/Smad 2 pathway (Wang et al., 2017), etc.
complements) leads to enhanced phagocytosis of nanomaterials
by MPS cells, thus resulting in their quick blood clearance and Biodegradation
accumulation in the liver and spleen (Cao et al., 2021; Stepien Following the exposure of nanomaterials, they may undergo
et al., 2018; Wang et al., 2019). For example, glucose-coated iron harsh and digestive gastrointestinal environments, microsomal
oxide nanoparticles (IONPs@Glc) are mainly accumulated in the enzymes in hepatocytes, as well as acidic, oxidative and ionic lyso-
liver and red pulp/marginal zone, whilst IONPs@PEG accumulated somes especially of the MPS systems, which challenge the integrity
homogenously across the liver and spleen and can be detected in of nanomaterials and contribute to their degradation (Cao et al.,
the reproductive organ, kidney, lung and heart after 72 h intra- 2021; Sohal et al., 2018; Vlasova et al., 2016). Depending on the
venous injection. This was attributed to that IONPs@Glc contained degradation degree and rate, and the toxic potential of intact nano-
more opsonins in their biocorona, whereas IONPs@PEG absorbed materials and degradation products, biodegradation has profound
a larger number of dysopsonins (Stepien et al., 2018). Moreover, effects on the toxicological profiles of nanomaterials (Cao et al.,
biocorona can impact the in vivo biodegradation of nanomaterials. 2021; Li, Liu et al., 2015; Li, Kroger et al., 2015 Li, Zhao et al.,
The same study reported that owing to the faster degradation of 2015; “Physical-Chemical Decision Framework to inform Decisions
absorbed albumin than that of fibrinogen, IONPs@PEG exhibited a for Risk Assessment of Manufactured Nanomaterials”; Wang et al.,
greater in vivo degradation degree than that of IONPs@Glc after 2015). For example, with a decrease in the ratio aspects of MSNs,
four months. In addition, the high uptake of opsonins-absorbed they possess a faster degradation rate in simulated gastric, intesti-
IONPs@Glc by macrophages resulted in the tightly packed lyso- nal, and body fluid, which was found to be responsible for the
somes and saturated enzyme activity, which also contributes to increased systemic absorption, liver distribution and more severe
the lower degradation of IONPs@Glc (Stepien et al., 2018). renal structural damage after oral administration (Li, Liu et al.,
At the cellular level, compared with bare nanomaterials, the 2015; Li, Kroger et al., 2015; Li, Zhao et al., 2015). In contrast
formed biocorona can greatly reduce the unspecific adhesion of to intravenously injected MSNs with similar ratio aspects, orally
nanomaterials onto cytomembrane, membrane integrity damage, delivered MSNs caused more serious kidney damage. This was pre-
and their cellular uptake efficiency and degree (Fig. 4(a)) (Lesniak sumably attributed to that MSNs in GIT undergo biodegradation
et al., 2013; Wang, Hartel, Ren, Graham, & Malmstadt, 2020). and may transform into some toxic chemicals and/or small irregu-
Despite the major negative effects of biocorona on cellular uptake, lar degradation particles thus eliciting severer kidney injury (Li,
specific proteins in biocorona with proper orientation can facilitate Liu et al., 2015; Li, Kroger et al., 2015; Li, Zhao et al., 2015). In
nanomaterial cellular uptake in various cells via receptor-mediated addition to affecting the toxicokinetics of nanomaterials, biodegra-
phagocytosis or endocytosis to some extend (Fig. 4(b)) (Liu, Tang, & dation is highly related to their toxicity mechanisms. The toxicity
Ding, 2020; Ritz et al., 2015). For instance, absorbed immunoglobu- mechanism of nanosilver has been validated to be owing to their
lins and complements may bind with Fc receptors and complement dissolution (Wang et al., 2015). In detail, Ag NPs in the form of
receptors respectively to trigger or enhance their phagocytosis by (Ag0 )n undergo dissolution in the acidic lysosome and turn into
MPS cells (Baimanov et al., 2020; Cai & Chen, 2018). Individual Ag+ , which leads to the enhanced LMP and thus the release of
Apo H-containing biocorona increased nanomaterial internaliza- lysosomal contents and Ag+ . Ag+ and lysosomal contents in the
tion by human mesenchymal stem cells, whereas Apo A4 or Apo C3 cytoplasm further damage mitochondrial, and result in ROS gener-
served as masks for unspecific cellular uptake (Ritz et al., 2015). ation and cell apoptosis. Upon dissolution in the lysosome, unstable
From above, biocorona presents complex effects on the cellular Ag+ ions evolve into Ag–O– presumably via binding with organic
uptake of nanomaterials, which is similar to their complicated acids. Once encountered with cytoplasm cysteine-contained pro-
impacts on nanotoxicity (Fig. 4(c)–(e)). In most cases, the for- teins, Ag–O– will become Ag–S– speciation, which may induce
mation of biocorona onto nanomaterials exerts mitigatory effects mitochondrion-involved apoptosis and facilitate silver excretion
on the unspecific deleterious effects of nanomaterials, which can (Fig. 5(a1 )–(a3 )) (Wang et al., 2015). However, biodegradation is not
be attributed to their capability of inhibiting cell binding and always tied to the negative effects of nanomaterials on biosystems.
uptake, reducing ROS generation, declining agglomeration rate, MoS2 nanosheets are promising for various biomedical applica-

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Y. Liu et al. Particuology 69 (2022) 31–48

Fig. 4. Biocorona greatly influences the cellular uptake and biological effects of nanomaterials. (a) The adsorption of nanoparticles into 293 T cells after 4 h incubation in FBS-
free culture media was reduced with the presence of protein corona. The scale bars are 30 ␮m (Wang et al., 2020). Copyright 2020, reprinted with permission from Royal Society
of Chemistry. (b) Various proteins in biocorona can facilitate the uptake of nanomaterials by cells especially macrophages via receptor-mediated endocytosis/phagocytosis
(Cai & Chen, 2018). Copyright 2018, reprinted with permission from John Wiley and Sons. (c) Biocorona can alleviate nanomaterial toxicity via several mechanisms (Cai &
Chen, 2018). Copyright 2018, reprinted with permission from John Wiley and Sons. (d–e) Biocorona aggravates nanomaterial toxicity via activating proinflammatory signaling
pathways (d) (Baimanov et al., 2020), Copyright 2020, reprinted with permission from American Chemical Society; or upregulating transforming growth factor (e) (Wang
et al., 2017), Copyright 2017, reprinted with permission from American Chemical Society.

tions due to their biocompatibility and versatile properties. Their monary lesions including granulomas, alveolitis, inflammation and
low toxicity and bioavailability have been demonstrated by many fibrotic responses (Muller et al., 2005).
studies (Cao et al., 2021; Xu, Zheng et al., 2020). Notably, Cao
et al. revealed that MoS2 @HSA nanocomplexes covered by protein
corona are mainly sequestered in the liver sinusoid and splenic red Analyzing approaches for nanotoxicology research
pulp, where they can transform into MoO4 2– facilitated by micro-
somal enzymes of hepatocytes, liver and splenic macrophages. The To achieve the safety evaluation of nanomaterials, it is important
molybdate degradation products do not cause obvious in vivo tox- to apply suitable analyzing approaches to obtain nanotoxicolog-
icity and finally be utilized for Moco biosynthesis in hepatocytes, ical profiles at the molecular, cellular and organ levels (Fig. 6).
which enhances the activities of molybdenum enzymes (Fig. 5(b)) Thorough characterization of nanomaterials is essential to gener-
(Cao et al., 2021). As for the majority of undegradable or extra- ate reliable and comparable research outcomes, find the causality
slow degradable nanomaterials, their biopersistence is responsible between nanomaterial physicochemical properties and their toxic-
for organ accumulation and potential long-term toxicity. For exam- ity, and conduct proper categorization or grouping for read-across
ple, CNTs with strong biodurability (pristine CNTs can stably exist of manifold nanomaterials (Faria et al., 2018; Leong et al., 2019;
for up to 24 months in vivo) have been of great concern (Lanone, Rasmussen et al., 2018). Since we focus on nano-bio interactions at
Andujar, Kermanizadeh, & Boczkowski, 2013; Liu et al., 2013). It varying levels, detailed methods for nanomaterial characterization
was reported that 80% CNTs were present as large agglomerates in are redirected to publications contributed by Mourdikoudis et al.
the lung following 60 days of inhalation, which caused severe pul- (Mourdikoudis, Pallares, & Thanh, 2018) and Kirsten Rasmussen
et al. (Rasmussen et al., 2018). Specifically, the latter is an outlook

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Y. Liu et al. Particuology 69 (2022) 31–48

Fig. 5. The biodegradation pattern of nanomaterials directly impacts their toxicity, underlying mechanism and bioavailability. (a1 ) Schematic diagram of Ag NPs’ degrada-
tion/transformation as the toxicity mechanism to human monocytes; (a2 ) Chemical transformation in silver species inside THP-1 cells indicated by silver K-edge XANES;
(a3 ) The spatial distribution of Ag NPs during endocytosis and exocytosis at the single-cell level (Wang et al., 2015). Copyright 2015, reprinted with permission from Amer-
ican Chemical Society. (b) Illustrations of the nano-bio interactions of MoS2 @HSA nanocomplex, their biodegradation and bioavailability (Cao et al., 2021). Copyright 2021,
reprinted with permission from Springer Nature.

based on OECD testing program, where methods for characterizing matrix (Qiu, Clement, & Haynes, 2018). Over the years, correla-
chemical composition, surface chemistry, size and size distribu- tion spectroscopy-based methods (e.g., fluorescence correlation
tion, surface area, Zeta potential, shape, porosity, crystalline phase, spectroscopy (FCS), scattering correlation spectroscopy and X-
hydrophobicity, dispersibility, agglomeration, redox potential, rad- ray photon correlation spectroscopy), surface-sensitive approaches
ical formation potential, and photocatalytic activity, as well as (e.g., surface-enhanced Raman spectroscopy (SERS) and imag-
future requirements were introduced (Rasmussen et al., 2018). Fol- ing surface plasmon resonance (SPR), attenuated total reactance
lowing, to study nanotoxicity under/near realistic scenarios, the Fourier transform infrared (ATR-FTIR), quartz crystal microbalance
formed/evolved biocorona, which acts as nanomaterials’ biologi- (QCM) and liquid NMR has been developed for in situ studying the
cal identity mediating the real nano-bio interactions, can not be binding or adsorption kinetics of biocorona in suspended bioflu-
ignored. Specific analytical methods for directly and/or indirectly ids (Bai et al., 2021; Carrillo-Carrion et al., 2017; Ge et al., 2015;
studying the identification, quantification, binding kinetics and Qiu et al., 2018). Notably, liquid NMR, SERS and ATR-FTIR give
structural information of biocoronal proteins have been summa- hints on not only the absorption kinetics but also the chemical
rized in these two reviews (Bai et al., 2021; Carrillo-Carrion, Carril, information of attracted proteins as well as their conformational
& Parak, 2017). Here, we briefly introduce the highly appreciated and structural changes (Mudunkotuwa, Minshid, & Grassian, 2014;
in situ biocoronal analyzing techniques (Fig. 6(a)–(e)), which pro- Qiu et al., 2018). Moreover, synchrotron radiation-based circular
vide real-time and realistic information of biocorona in biological dichroism(SR-CD) with high light flux in ultraviolet regions allows

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Fig. 6. The integration of analytical methods for studying nano-bio interactions at different levels. (a) SPR as an indirect method for biocorona analysis (Carrillo-Carrion et al.,
2017). Copyright 2017, reprinted with permission from Elsevier. (b) ATR-FTIR spectra for adsorbed glycine (0.5 mM) on TiO2 nanoparticles were recorded as a function of
time after the initial introduction of each into the ATR-FTIR flow cell (Mudunkotuwa et al., 2014). Copyright 2014, reprinted with permission from Royal Society of Chemistry.
(c) QCM-D spectra showing frequency change as a function of time of fibrinogen adsorption on smooth and nanotopography modified surfaces (Visalakshan, Cavallaro et al.,
2019). Copyright 2019, reprinted with permission from John Wiley and Sons. (d) FCS as an indirect method for analyzing nanomaterial-protein interaction (Carrillo-Carrion
et al., 2017). Copyright 2017, reprinted with permission from Elsevier. (e) SR-CD spectra of PBS-suspended HSA before and after introducing with NSs (Baimanov et al., 2020).
Copyright 2020, reprinted with permission from American Chemical Society. (f) Top view of the internalization pathway for cubic PEGylated NPs with grafting density 0.6
chains/nm2 (t = 0.24 s) (Li, Liu et al., 2015; Li, Kroger et al., 2015). Copyright 2015, reprinted with permission from Royal Society of Chemistry. (g) TEM images of cells and Ag
NPs (Wang et al., 2015). Copyright 2015, reprinted with permission from American Chemical Society. (h) Effect of the protein corona on cellular adhesion of nanoparticles
and cell viability (Wang et al., 2020). Copyright 2020, reprinted with permission from Royal Society of Chemistry. (i) STXM dual energy contrast images of Gd@C82 (OH)22
internalized by a primary mouse peritoneal macrophage in vivo (Chen et al., 2014). Copyright 2014, reprinted with permission from John Wiley and Sons. (j) Changes in
silver chemical species of the cell samples during the cellular uptake of Ag NPs and the removal of silver according to silver K-edge XANES (Wang et al., 2015). Copyright
2015, reprinted with permission from American Chemical Society. (k) Flow cytometry-based assay for the detection of exposed TGF-␤1 in the corona (Wang et al., 2017).
Copyright 2017, reprinted with permission from American Chemical Society. (l) Multi-omics analysis as one of the possible toxicity testing techniques used in risk assessments
of nanomaterials (Chen et al., 2018). Copyright 2018, reprinted with permission from Springer Nature. (m–o) ICP-MS as element-based method and SPECT/CT and MRI as
imaging-based methods for quantifying in vivo distribution of nanomaterials (Wang et al., 2018). Copyright 2018, reprinted with permission from American Chemical Society.
(p) LA-ICP-MS gold images showing transportation and intra-organ distribution of PEG-GNPs in the liver after 24 h intravenous injection (Li, Zhang et al., 2020; Li, Wang et al.,
2020). Copyright 2020, Li et al.; open access. (q) Mapping of molybdenum in the liver by SR-XRF microscopy (Cao et al., 2021). Copyright 2021, reprinted with permission
from Springer Nature. (r) Molybdenum K-edge XANES (solid lines) and fitted curves (dashed lines) of livers from BALB/c mice at the indicated times post injection for studying
the in vivo biotransformation of MoS2 (Cao et al., 2021). Copyright 2021, reprinted with permission from Springer Nature.

for accurate and trace measurement of the secondary structures microscopy ((S)TXM), X-ray diffraction microscopy (XDM), nano-
of biocoronal proteins, their conformational changes and dynam- CT; transmission electron microscopy (TEM), scanning electron
ics even in the presence of other substances (Chen, Li, Qu, Chai, & microscopy (SEM); secondary ion mass spectroscopy (SIMS) and
Zhao, 2013). In addition, some of the above experimental methods atomic force microscopy have been utilized for visualizing nano-
such as SPR and QCM and computional simulation (Fig. 6(f)) can be material cellular uptake, intracellular localization and cellular
used to study the interactions of nanomaterials with other kind of structural damages (Behzadi et al., 2017; Chen et al., 2014; Li,
biomolecules such as lipid (Li, Liu et al., 2015; Li, Kroger et al., 2015; Liu et al., 2015; Li, Kroger et al., 2015; Li, Zhao et al., 2015; Qiu
Li, Zhao et al., 2015). et al., 2018; Wang et al., 2015). Notably, SR-facilitated (S)TXM with
Furthermore, nanomaterial risk assessment demands a deep large focus depth and strong penetration allows for 3D single-cell
understanding of their cellular uptake, trafficking and toxicity tomography and detecting element-specific distribution at even
mechanisms (Fig. 6(g)–(l)). Various light, X-ray and elec- single-particle level (Li, Liu et al., 2015; Li, Kroger et al., 2015;
tron microscopy-based and MS imaging techniques including Wang et al., 2015). SIMS techniques detecting the ejected secondary
light/dark-field microscopy, confocal laser scanning microscopy ions after laser sputtering samples can visualize the intracellular
(CLSM), structural illumination microscopy (SIM), stochastic opti- localization and chemical transformations of nanomaterials at the
cal reconstruction microscopy (STORM); (soft) transmission X-ray single-cell level (Wang et al., 2018). In addition, super-resolution

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Y. Liu et al. Particuology 69 (2022) 31–48

microscopy (e.g., SIM, STROM) and X-ray-based microscopy (e.g., capability (Li, Liu et al., 2015; Li, Kroger et al., 2015; Li, Zhao et al.,
TXM and XDM) both with resolution limit at tens of nanometers, 2015). SR-X-ray fluorescence imaging (SR-XRF) can map and semi-
and electron microscopy with resolution limit at few nanometers quantify multiple elements in complex biological matrices, thus
are capable of conducting sing-cell imaging of nanomaterials (Qiu indicating the absorption and excretion kinetics, and sub-organ dis-
et al., 2018). Single-cell analysis is highly desirable to investigate tribution patterns of nanomaterials (Cao et al., 2021; Wang et al.,
the real cellular response and establish dose-effect relationships for 2018). SR-X-ray absorption near-edge structure (SR-XANES) can
nanomaterials, due to its merits in eliminating the heterogeneity of in situ indicate alterations in the chemical or electronical state of
nanomaterials and cell population in terms of genetic variation, bio- elements to reveal the metabolism or biotransformation of nano-
chemical state, nanoparticle uptake, etc (Qiu et al., 2018). Another materials at even single-particle level (Cao et al., 2021; Wang et al.,
category for single-cell analysis is cytometry-based techniques 2018, 2015). By combining SR-XRF and SR-XANES, a complete
including flow cytometry and mass cytometry, which can reveal picture of the in situ distribution and transformation of nanoma-
nanomaterials’ cellular uptake, toxic effects, alterations in cellular terials can be obtained (Wang et al., 2018). What’s more, another
biochemical state and phenotypic, etc. in a high throughout and group of state-of-art techniques for exploring nanomaterial distri-
multiparameter manner via analyzing forward- and side-scattered bution and metabolism/speciation are MS-based techniques. Laser
light, fluorescent and atomic mass spectrometric (Bendall et al., ablation ICP-MS (LA-ICP-MS) and matrix-assisted laser desorp-
2011; Lesniak et al., 2013; Qiu et al., 2018; Wang et al., 2015). tion/ionization MS (MALDI-MS) utilize laser beams to vaporize
As for exploring the cytotoxicity and underlying mechanisms of and ionize the surface of samples respectively and then coupled
nanomaterials, a wide range of assays and techniques detecting with MS analysis, which allows for in situ quantifying the biodis-
cell viability, cell proliferation, cell metabolic activity, genotoxic- tribution of nanomaterials in tissue slices (Li, Zhang et al., 2020;
ity, necrosis and apoptosis, membrane integrity, ROS generation, Li, Wang et al., 2020; Qiu et al., 2018; Wang et al., 2018). More-
gene expression of proinflammatory cytokines, receptors and sig- over, the combination of HPLC with ICP-MS permits the efficient
naling molecules, etc. are commonly applied (Khlebtsov & Dykman, separation and accurate quantification of manifold nanomaterials’
2011; Magdolenova et al., 2014; Murugadoss et al., 2020; Wu, Jiang metabolites with distinct chemical composition and coordination.
et al., 2018; Wu, Shan et al., 2018; Xia et al., 2008). However, Single-particle ICP-MS (SP-ICP-MS) is able to quantify the number
these conventional approaches only offer isolated and empirical concentration and size changes of nanomaterials and their exist-
cytotoxicity and mechanism outcomes, for a more integrated and ing forms as dissolved ions or particles in extracted tissue samples
holistic understanding of nanotoxicity, systems toxicology rely- to reflect theirs in vivo metabolism (Rasmussen et al., 2018; Wang
ing on multi-omics analysis is highly appreciated (Costa & Fadeel, et al., 2018). To summarize, Table 1 lists the related advanced ana-
2015). Systems nanotoxicology focuses on the changes of gene, lytical approoaches for studing nano-bio interactions at molecule,
mRNA, proteins and metabolites after nanomaterial perturbation, cell and organ levels and compares their advantages and limiations.
which screens for multiple end-points in a single analytical run.
Nevertheless, how to link the subtle molecular alterations in the
results of transcriptomics, proteomics, metabolomics, genomics, Regulation
and epigenomics with cytotoxicity phenotypes is very challenging
but pivotal (Costa & Fadeel, 2015). As discussed above, the toxicity of nanomaterials is closely
At the animal level, the toxicokinetics of nanomaterials, which associated with their unique physicochemical properties, some
answer how they are absorbed into, distributed in, metabo- of which however endow nanomaterial-containing products with
lized by and eliminated from the body should be conducted more promising performances and wider applications than that
(Fig. 6(m)–(r)) (Wang et al., 2018). Conventional strategies for of traditional substances. The so-called nanomaterial paradox has
absorption, distribution and elimination research are mainly to created difficulties for stakeholders in developing and harnessing
conduct time-course analysis of the concentration of nanomate- nanotechnology without causing negative implications. To tackle
rials in the blood, digested tissue samples dissected from organs of this problem, important regional and national regulatory bodies
interest, and urine/feces (Kreyling, Holzwarth, Haberl et al., 2017; have been taking active action on the supervision of nano-enabled
Li, Zhang et al., 2020; Li, Wang et al., 2020; Wang et al., 2018). products and cooperating to establish global standardization and
Analyzing techniques are dependent on the nature of the tested regulation consensus (Hamburg, 2012; Park & Yeo, 2016; Rauscher,
nanomaterials, for example, inductively coupled plasma (ICP)- Rasmussen, & Sokull-Klüttgen, 2017). At the present stage, the
optical emission spectrophotometry (OES), ICP-mass spectrometry regulation of nanomaterial-containing products is covered by the
(MS), atomic absorption spectrophotometry (AAS) for quantifying existing general and sector-specific regulatory and legislation sys-
metal element contained nanomaterials (Chen et al., 2013; Das tems (Hamburg, 2012; Soltani & Pouypouy, 2019). For instance, in
et al., 2017; Mourdikoudis et al., 2018; Pauluhn, 2010; Wang et al., the European Union (EU), nanomaterials are under the same regu-
2018), isotopic tracing (IT) for radiolabeled-nanomaterials (Wang latory framework as all chemicals and mixtures, i.e., nanomaterials
et al., 2018), 1 H-NMR for 14 C-labeled polymeric nanoparticles have to be registered by the Registration, Evaluation, Authorization,
(Bertrand et al., 2017), fluorescence spectrophotometry for fluores- and Restriction of Chemicals regulations before their import and
cent nanomaterials (Meng, Wang, Ping, & Yeo, 2018), etc. Moreover, circulation in the EU; nanomaterials with hazardous properties are
several in vivo imaging techniques have been adopted for real-time, obligated to notify to the European Chemicals Agency and should
non-invasive, and high sensitive toxicokinetics analysis, which be labeled and packaged according to the Classification, Labelling
include positron emission tomography (PET), single-photon emis- and Packaging Regulation regulations to ensure their safe use
sion computed tomography (SPECT), computerized tomography (“Regulation of nanomaterials in the European Union Observatory
(CT), magnetic resonance imaging (MRI), two-photon fluorescence for nanomaterials (EUON)”). Moreover, based on the report titled
imaging (FI), and photoacoustic imaging (PAI), etc (Liu, Wang, “Second Regulatory Review on Nanomaterials” launched by the
Zhang, Wang et al., 2016; Liu, Wang, Zhang, Wu et al., 2016; Wang European Commission, “possible risks are related to specific nano-
et al., 2018; Zhang et al., 2020; Chen et al., 2013). Noteworthy, syn- materials and specific uses. Risk assessment of nanomaterials
chrotron radiation (SR)-based X-ray techniques are highly desirable should be performed on a case-by-case basis, using pertinent infor-
for in situ exploring the biodistribution and metabolism of nanoma- mation” (“Second Regulatory Review on Nanomaterials”). In this
terials in biological samples with high sensitivity, deep penetration, regard, there are corresponding guidance documents established
favorable spatial and temporal resolution and elemental mapping by organizations such as the European Food Safety Authority, Euro-

43
Y. Liu et al. Particuology 69 (2022) 31–48

Table 1
List of advanced analytical techniques for different aspects of nanotoxicological research.

pean Medicines Agency, Joint Research Centre-Institute for Health Products”; Hamburg, 2012). Whether a product is subject to pre-
and Consumer Protection, and European Agency for Safety and market review (e.g., new drugs and biological products) or not
Health at Work, which aims to facilitate the regulation of nanoma- (e.g., cosmetics), the industry is responsible for ensuring that the
terials in novel foods, food contact materials, cosmetic products, product meets the applicable safety standards and other applica-
medical devices and enable consumer, worker and environmen- ble requirements (Hamburg, 2012). Additionally, the FDA offers
tal protection (“Regulation of nanomaterials in the European general and specific technical guidances covering different topics
Union Observatory for nanomaterials (EUON)”; Soltani & Pouypouy, and encourages early industry consultation to help the indus-
2019). Similarly, in the United States (US), the Food and Drug try meet its statutory obligations (“FDA’s Approach to Regulation
Administration (FDA) as one of the most important regulatory of Nanotechnology Products”; Hamburg, 2012). Another impor-
institutions, maintains its product-focused regulatory policies for tant US governmental agency for supervising nanomaterials is
nano-enable products and regulates them under existing statutory the Environmental Protection Agency, which launched a series of
authorities via specific premarket review and/or postmarket over- environmental regulations including the Toxic Substances Con-
sight systems (“FDA’s Approach to Regulation of Nanotechnology trol Act, Federal Insecticide, Fungicide, and Rodenticide Act, Clean

44
Y. Liu et al. Particuology 69 (2022) 31–48

Air Act, and Clean Water Act, etc. to regulate nanomaterials dur- Declaration of interests
ing their manufacture, use, distribution in commerce, and disposal
and their release into the environment (Hanson, Harris, Kalpana, The authors declare that they have no known competing finan-
Ramakrishnan, & Thompson, 2011). In other countries, nanomate- cial interests or personal relationships that could have appeared to
rials and ordinary chemicals are ruled by shared legal acts for their influence the work reported in this paper.
regulation, such as Act on the Evaluation of Chemical Substances
and Regulation of Their Manufacture, Food Sanitation Act, and Declaration of Competing Interest
Pharmaceutical and Medical Device Act, etc. in Japan, and Quality
Management & Safety Control of Industrial Products Act, Industrial The authors report no declarations of interest.
Safety & Health Act, and Toxic Chemical Control Act, etc. in Korea
(Rasmussen et al., 2017). To summarize, although nanomaterials
share similar regulation and legislation frameworks to ordinary Acknowledgements
chemicals in the current state, almost all regulatory agencies pay
special attention to and launch guidances or standardizations for This work was supported by the Strategic Priority Research Pro-
almost every stage of the safety evaluation of nanomaterials. There gram of Chinese Academy of Sciences (grant No. XDB36000000);
are also advocators appealing to construct legislative and regula- the National Basic Research Program of China (grant No.
tory frameworks exclusively for nanomaterials. It is believed that 2020YFA0710702); the National Natural Science Foundation of
the regulation and legislation for nanomaterials will be further China (grant Nos. 51822207 and 51772292).
completed with the development of risk assessment of nanoma-
terials (Rasmussen et al., 2017). References

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