Nanotechnologies -- High throughput screening method for nanoparticles toxicity using 3D model cells

This document describes a method for high throughput evaluation of cytotoxic response of 3D model cells exposed to NPs without optical interference. The method in this document is intended to be used in biological testing laboratories that are competent in the culture and growth of cells and the evaluation of cytotoxicity of NPs using 3D-model cells. This method applies to materials that consist of nano-objects such as nanoparticles, nanopowders, nanofibres, nanotubes, and nanowires, as well as aggregates and agglomerates of these materials.

Nanotechnologies -- Méthode de criblage à haut débit de la toxicité des nanoparticules utilisant des systèmes cellulaires 3D

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TECHNICAL ISO/TR
REPORT 22455
First edition
2021-11
Nanotechnologies — High throughput
screening method for nanoparticles
toxicity using 3D model cells
Nanotechnologies — Méthode de criblage à haut débit de la toxicité
des nanoparticules utilisant des systèmes cellulaires 3D
Reference number
ISO/TR 22455:2021(E)
© ISO 2021
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ISO/TR 22455:2021(E)
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© ISO 2021

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© ISO 2021 – All rights reserved
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ISO/TR 22455:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Background ............................................................................................................................................................................................................... 2

4.1 General ........................................................................................................................................................................................................... 2

4.2 Effects of optical properties of NPs on in vitro cell viability assays ...................................................... 2

4.3 New assay platform for in vitro toxicity screening of NPs diminishing optical

interference ............................................................................................................................................................................................... 4

4.4 Characteristics of 3D model cells ........................................................................................................................................... 7

4.5 Cell viability in response to NPs assessed using 3D model cells on a pillar insert .................... 9

4.6 Cellular uptake of NPs using 3D model cells on a pillar insert .................................................................13

4.7 Discussion of alternative strategies to evaluate in vitro toxicity testing of NPs ..................... 16

5 Methods for cell viability screening of NPs using 3D-model cells ................................................................17

5.1 General ........................................................................................................................................................................................................ 17

5.2 Cell culture .............................................................................................................................................................................................. 17

5.3 Preparation of the pillar insert for in vitro screening ..................................................................................... 17

5.4 Encapsulation of cells on a micropillar chip to generate 3D-model cells ........................................ 18

5.5 NPs sample preparation .............................................................................................................................................................. 18

5.6 Exposing 3D-model cells to NPs ........................................................................................................................................... 18

5.7 Cell viability analysis using a WST assay ..................................................................................................................... 19

5.8 Cell viability analysis using live-cell imaging .......................................................................................................... 19

Bibliography .............................................................................................................................................................................................................................21

iii
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ISO/TR 22455:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2021 – All rights reserved
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ISO/TR 22455:2021(E)
Introduction

With an increasing number of nano-products including nanoparticles (NPs), potential exposure of

consumers to NPs has increased. Therefore, the human and environmental impacts of NPs have recently

emerged as an issue. High-throughput screening (HTS) approaches are often used for NPs toxicity

screening. However, there are still limitations to provide the reproducible and reliable results based on

a HTS method. To assess the potential toxicity of manufactured or engineered NPs, traditional in vitro

toxicity studies have been performed using a surface attached two-dimensional (2D) culture system.

2D assays for cellular metabolic activity, cytotoxicity, or oxidative stress have been widely used in the

first stage of hazard evaluation. However, several problems were encountered during assay validation,

ranging from particle agglomeration in biological media to optical interference with the assay platform.

There are ISO documents on the cytotoxic effects of NPs using cell viability assays and detection

of reactive oxygen species (ROS) levels, but they can be applicable for a few classes of NPs that are

well-dispersed in the media. Additionally, reagents used in the assays can interact with tested NPs or

interfere with spectrophotometric reading.

This document describes a new assay platform, consisting of three-dimensional (3D) arrangement of

cells on pillar inserts to evaluate cell viability and diminish artefacts arising from optical interferences

and NP reactivity with assay components.

This document aims to overcome the optical interference of NPs and obtain reliable and reproducible

cell viability results. The 3D-model cells are exposed to fresh cell viability reagent by simply transferring

and immersing the pillar insert from one well to another well without optical interference from the

NPs. In addition, 3D-model cell culture approaches facilitate cell-cell interactions and enhance cell-to-

cell or cell-to-extracellular matrix (ECM) adhesion/signalling, ultimately leading to the expression of

phenotypic proteins/genes and the formation of in vivo tissue-like morphology. It generates uniform

cell-containing hydrogel droplets on the pillar insert and allows to easily change cell growth media

or expose 3D-model cells to analytical reagents by immersing the tip of the pillar insert in different

reaction plates.
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TECHNICAL REPORT ISO/TR 22455:2021(E)
Nanotechnologies — High throughput screening method
for nanoparticles toxicity using 3D model cells
1 Scope

This document describes a method for high throughput evaluation of cytotoxic response of 3D model

cells exposed to NPs without optical interference.

The method in this document is intended to be used in biological testing laboratories that are competent

in the culture and growth of cells and the evaluation of cytotoxicity of NPs using 3D-model cells.

This method applies to materials that consist of nano-objects such as nanoparticles, nanopowders,

nanofibres, nanotubes, and nanowires, as well as aggregates and agglomerates of these materials.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 and the following

apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
agglomerate

collection of weakly bound particles or aggregates or mixtures of the two where the resulting external

surface area is similar to the sum of the surface areas of the individual components

Note 1 to entry: The forces holding an agglomerate together are weak forces, for example, van der Waals forces,

or simple physical entanglement.

Note 2 to entry: Agglomerates are also termed secondary particles, and the original source particles are termed

primary particles.

[SOURCE: ISO/TS 80004-2:2015, 3.4, modified — "weakly or medium strongly bound particles" has

been replaced with " weakly bound particles or aggregates or mixtures of the two".]

3.2
dispersion

microscopic multi-phase system in which discontinuities of any state (solid, liquid or gas: discontinuous

phase) are dispersed in a continuous phase of a different composition or state

Note 1 to entry: If solid particles are dispersed in a liquid, the dispersion is referred to as a suspension. If the

dispersion consists of two or more liquid phases, it is termed an emulsion. A super emulsion consists of both solid

and liquid phases dispersed in a continuous liquid phase.
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ISO/TR 22455:2021(E)
[SOURCE: ISO 19007:2018, 3.2]
3.3
nano-object
material with one, two or three external dimensions in the nanoscale (3.5)
Note 1 to entry: This is a generic term for all discrete nanoscale objects.

[SOURCE: ISO/TS 80004-2:2015, 2.2, modified — "discrete piece of" has been added to the definition

and Note 1 to entry has been replaced.]
3.4
nanoparticle
nano-object (3.3) with all three dimensions in the nanoscale (3.5)

Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically by

more than three times), the terms nanorod or nanoplate are intended to be used instead of the term nanoparticle.

3.5
nanoscale
size range from approximately 1 nm to 100 nm

Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be

exhibited in this size range. For such properties, the size limits are considered approximate.

Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small

groups of atoms from being designated as nano-objects (3.3) or elements of nanostructures, which can be implied

by the absence of a lower limit.
3.6
high-throughput screening

method that comprises the screening of a large number of chemicals via automation, miniaturized

assays and large-scale data analysis

Note 1 to entry: This protocol can be applied to screen the toxicity of NPs based on 96-well plate or 532 microchip.

4 Background
4.1 General

With the increase in the number of consumer products containing NPs, potential exposure to NPs has

increased, and potential human and environmental hazards of NPs have emerged. To assess the effects

of NPs, a high-throughput screening method to evaluate cell viability following exposure to NPs is

[1]

needed. High-throughput approaches have been used to screen for toxicity of manufactured NPs .

4.2 Effects of optical properties of NPs on in vitro cell viability assays
[2][3]

NPs possess linear/nonlinear optical absorbance and photoluminescence emission . Because of

their physicochemical and optical properties, NPs are used in various fields for disease diagnoses or as

industrial products. Most NPs exhibit the optical properties in a wide range of absorbance wavelengths,

where optical interference can be pronounced in cell viability assays based on absorbance read-outs.

To assess the potential toxicity of manufactured or engineered NPs, traditional in vitro toxicity studies

have been performed using 2D model culture systems. During validation of 2D-model assays, several

problems, for example, particle agglomeration in biological media and optical interference with the

[4][5]

assay system, were encountered . In traditional cell viability assays, colorimetric detection is

generally used, and luminescent and fluorescent detection methods have been also applied to evaluate

the cell viability assay. As shown in Table 1, some NPs such as the Ag NPs show an optical absorption at

the wavelengths where the colorimetric assays are monitored.
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ISO/TR 22455:2021(E)
Table 1 — Range of the wavelengths of conventional cell viability assays
Assay Wavelength used for measurement
WST-1 420 to 480
XTT 450 to 500
WST-8 450 (450 to 490)
MTS 490 (450 to 540)
NRU 540
MTT 570 (500 to 600)

Representative cell viability assays include water-soluble tetrazolium (WST), 2,3-Bis-(2-methoxy-4-

nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), neutral red uptake (NRU), and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays as well as ATP detection based

on luminescence, but the optical properties of NPs can influence all of these detection methods. The

effects of the optical absorbance of NPs (Ag, silver-shelled gold (Au@Ag), and iron (II, III) oxide (Fe O )

3 4

NPs and single-wall carbon nanotube (SWCNT) on cell viability were evaluated as shown in Figure 1,

a). The absorbance increased according to the concentration of NPs. Although some remedies, such as

multiple washing steps, NPs attached to the cultured cells can still influence the optical absorption

[6]

reading. These optical properties can lead to false positive or false negative results . The luminescence

detection method is also vulnerable to the optical interference of NPs, as shown in Figure 1, b). As the

number of NPs increased, the intensity of luminescence remarkably decreased, showing false positive

cytotoxicity.
a) Colorimetric detection using CCK-8 (Cell Counting Kit-8) reagent (Dojindo)

CCK-8 (Cell Counting Kit-8) reagent (Dojindo) is an example of a suitable product available commercially. This information is

given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.

CCK-8 (Cell Counting Kit-8) reagent (Dojindo) is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO of this product.

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ISO/TR 22455:2021(E)
b) Luminescence detection using CellTiter-Glo® reagent (Promega)

CellTiter-Glo® reagent (Promega) is an example of a suitable product available commercially. This information is given for

the convenience of users of this document and does not constitute an endorsement by ISO of this product.

Key
X concentration (µg/ml) 1 Ag
Y1 cell viability (%) 2 Ag-shelled Au
Y2 absorbance 3 Fe O
3 4
Y3 luminescence 4 SWCNT
5 Au
cell viability
6 QD
background absorption
background values
Figure 1 — Effects of optical properties of NPs on cell viability

A549 cells were treated with NPs under the pre-dose finding ranges for 24 h, and cell viability was

measured using the colorimetric and luminescence detection method. The absorbance of NPs without

cells was measured as the background. The left axis represents the relative cell viability, and the right

axis represents the absorption values at 450 nm (A) and luminescence (B). For colorimetric detection

using CCK-8 (Cell Counting Kit-8) reagent (Dojindo) in panel (A), the cell viability and background

absorption are represented with bars and dashed lines respectively. For luminescence detection using

CellTiter-Glo® reagent (Promega) in panel (B), the cell viability and background values are represented

with bars and dashed lines respectively. SWCNT, single-walled carbon nanotube; QD, quantum dot.

4.3 New assay platform for in vitro toxicity screening of NPs diminishing optical

interference

3D-model cells on a pillar insert are used to evaluate the cell viability while minimizing artefacts such

as those associated with optical absorption and undesirable reactions with an assay reagent. In the use

of these platforms, the 3D-model cells are exposed to fresh cell viability reagent by simply transferring

and immersing the pillar insert from a column of a well to another column of that well without the

optical interference of the NPs, and the schematic flow is shown in Figure 2. This platform allows to

easily exchange cell growth media and to expose the 3D-model cells to analytical reagents by immersing

CellTiter-Glo® reagent (Promega) is an example of a suitable product available commercially. This information is given for the convenience of

users of this document and does not constitute an endorsement by ISO of this product.

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ISO/TR 22455:2021(E)
[7][10]

the tip of the pillar insert in different reaction plates . Thus, this method allows high-throughput

screening of NPs cytotoxicity by reducing optical interference and reactivity with assay reagents.

Key
1 pillar insert 8 PBS
2 96-well 9 reagent
3 growth medium 10 absorbance
4 nanoparticles 11 fluorescent intensity
5 cell seeding 12 washing
6 cell culture 13 assay
7 treatment 14 measurement of cell viability (10 or 11)

Figure 2 — Schematic flow of the pillar insert system for evaluating the cell viability of NPs

The cell-alginate mixture was dispensed onto a pillar insert. The encapsulated 3D cells on the pillar

insert were cultured in the medium and then exposed into NPs. The following exposed 3D cells on pillar

insert is easily transferred into an independent well with WST-8 or calcein AM reagent and the cell

viability can be measured by absorbance or fluorescence.

The method is applicable to 96-well plates and can be extended to 532-well plates microchips (see

Figure 3).
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ISO/TR 22455:2021(E)
a) 96-well system
b) 532-well microchip system
Key
1 8-pillar strip 8 slide glass (75 mm × 25 mm)
2 96-pillar plate 9 top micropillar
3 pillar insert 10 bottom microwell
4 96-well plate 11 pillar
5 pillar insert 12 cells in alginate
6 micropillar chip 13 well
7 microwell chip 14 stamping
NOTE Each pillar insert was immersed into 96 wells or 532 microwells.

Figure 3 — Schematics of the assay platform to screen the cell viability of NPs based on a 96-

well or 532-well microchip system
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ISO/TR 22455:2021(E)
4.4 Characteristics of 3D model cells

Owing to the lack of physiological functions in vivo in traditional 2D culture models, 3D cells have been

[11]

described as more predictive in hazard evaluation . Advantages and challenges for evaluating the

[12]

toxicity of NPs have been discussed between 2D and 3D culture models . A plastic pillar insert is

used to facilitate miniaturized 3D cell culture in a 96-well plate and 532-well microchip by forming 3D

hydrogel droplets containing cells on the tip of the pillar insert. 2D cell cultures have been generally

used previously in toxicity studies, but many cells of normal and malignant origin lose some of their

[13][14]

phenotypic properties when grown in vitro as a 2D monolayer . The formation of tissue-like

structures is highly inhibited in a 2D monolayer culture due to the strong affinity of cells for most

artificial surfaces and the restriction to a 2D space, which severely limits intercellular contacts and

interactions. In addition, 3D-model cell culture approaches facilitate cell-cell interactions and cell-to-

cell or cell-to-ECM adhesion/signalling, ultimately leading to the expression of phenotypic proteins/

[15]-[18]

genes and the formation of in vivo tissue-like morphology . It is evident that a 3D model restores

the morphological and functional characteristics of tissues. For instance, cytoskeletal structure or ECM

[18]

adhesion molecules are more similar to in vivo features in 3D fibroblasts . 3D A549 also showed the

increased expression of structural and functional markers including tight junction, epithelial proteins

[15]

and mucin-specific proteins . The previous investigations demonstrated that the toxic effects of NPs

[16]

in 3D cell model correlated well with the animal study data . To demonstrate the feasibility of pillar

insert for 3D cell culture, A549 and PC9 cell lines from human non-small cell lung cancer (NSCLC) were

grown on the pillar insert, and their 3D morphology, growth rate and cytotoxicity (with Erlotinib) were

tested, as shown in Figure 4. The dispensed single cells in alginate matrix grew on the pillar insert,

forming a unique 3D structure by cell-cell and cell-ECM interaction during proliferation [see Figure 4,

a)]. The increase of green fluorescence and morphological observation over time also evidenced

the growth of 3D A549 cells [see Figure 4, b)]. The number of cells in alginate droplets was linearly

proportional to the cell seeding density, indicating the uniformity of encapsulated 3D cells on the pillar

insert [see Figure 4, c)]. As a proof of concept showing the functionality of 3D cells, the cytotoxic effects

of Erlotinib, an inhibitor of epidermal growth factor receptor (EGFR) on PC9 cells (EGFR mutant type)

and A549 (EGFR wild type) were compared between 2D and 3D culture system. PC9 cells with EGFR

mutation are known to show a great response to Erlotinib (i.e. extremely low half-maximal inhibitory

[19]

concentration, C values were obtained) . Interestingly, the C value of 3D PC9 cells grown on

i,50, i,50

the pillar insert was six times lower than those obtained from 2D PC9 cells, whereas the C value

i,50

of 3D A549 cells were five times higher than those from 2D A549 cells [see Figure 4, d) and Figure 4,

e)]. This kind of disparity on C values between 2D and 3D cell culture systems has been reported

i,50
[20]

in the literature and is considered as a distinctive characteristic of cells cultured in 3D system

[21]

. The result can represent that A549 and PC9 cells grown on the pillar insert better mimic tissue

environment.
NOTE A549 cells were stained with calcein AM.

a) Alginate droplet containing A549 cells on the tip of the pillar inserts and A549 cells forming

3D structures in the droplet
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ISO/TR 22455:2021(E)

b) Pictures of stained A549 cells in alginate on the tip of the pillar insert after 1-day and 4-day

incubations

NOTE The linear increase in absorbance indicates that a uniform number of cells are encapsulated in

alginate droplets.

c) Absorbance of A549 cells encapsulated in alginate on the tip of the pillar inserts at different

seeding densities measured by the CCK-8 reagent

d) Dose-response curves of Erlotinib with A549 and PC9 cells cultured in 3D alginate droplets

on the pillar inserts and 2D monolayers

e) Comparison of the doubling times of A549 and PC9 cells cultured in 2D monolayers and 3D

alginate droplets and their C values obtained with Erlotinib
i,50
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ISO/TR 22455:2021(E)
Key
X1 # of A549 cells (1 000 cells) 3 day 1
X2 # of PC9 cells (1 000 cells) 4 day 4
X3 log [erlotinib (pM)]
A549_2D
Y1 absorbance
PC 9_2D
Y2 live cell (%)
A549_3D
1 pillar insert
PC9_3D
2 3D cultured A549
SOURCE: Reference [7], reproduced with the permission of the authors.
[ ]
Figure 4 — Characteristics of 3D-model cells on pillar insert 7

4.5 Cell viability in response to NPs assessed using 3D model cells on a pillar insert

The cell viability of NPs with or without optical interference was evaluated in both 2D monolayer and

3D-model cells on pillar insert. At first, the applicability of the pillar insert system for cell viability

assay was tested with NPs showing no significant optical interference. The 70-nm silica (SiO ) NPs and

QD selected as negative and positive reference NPs for cytotoxicity and the cell viability was compared

between the 2D monolayer and 3D-model cells on pillar insert (see Figure 5). The cell viability of

negative SiO NPs and positive QD were similar between both culture systems, indicating that cell

viability assay is applicable in the pillar insert system.
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ISO/TR 22455:2021(E)

a) Measurement of cell viability of 70 nm SiO in 2D monolayer and 3D model A549 cells

b) Measurement of cell viability of QD in 2D monolayer and 3D model A549 cells
Key
X1 70 nm SiO (µg/ml) 1 2D monolayer
Y1 cell viability (%) 2 3D culture on pillar insert
X2 QD (µg/ml)
CCK-8
Y2 absorbance
NPs only

Figure 5 — Cell viability of NPs without optical interference in 2D and 3D culture systems

A549 cells were cultured in a conventional 2D monolayer and as 3D-model cells on a pillar insert.

Well-dispersed 70 nm SiO NP and QD were selected as negative and positive reference NP to test

the cytotoxicity, respectively. The cell viability was compared between the two systems after NPs

treatment for 24 h. The pre-range dose finding test was performed to determine the concentration at

which cell viability is reduced for both negative and positive NPs. Alternative concentration can be used

depending on dispersion or precipitation. Cell viability was measured using a WST-8 (CCK-8) assay.

On the other hand, the cell viability of Ag NPs showing optical interference was compared in both culture

system using a WST-8 assay (Figure 6). The size of the Ag NPs was certified by the manufacturer to be

7,9 nm ± 0,95 nm based on transmission electron microscopy (TEM) and 11,5 nm ± 10,5 nm based on

dynamic light scattering (DLS). The Ag NPs showed a unique absorbance with a maximum at 410 nm.

Light microscopy showed that many Ag NPs were attached to HepG2 cells. In the 2D culture platform,

cell viability differs dependin
...

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