Nanotechnologies — Method to estimate cellular uptake of carbon nanomaterials using optical absorption

This document describes a near-infrared optical absorption method to estimate the in vitro cellular uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake; additional analysis using a different technique can be required if quantification is desired.

Nanotechnologies — Méthode d'estimation de la captation cellulaire des nanomatériaux carbonés par mesure d'absorption optique

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Publication Date
04-Aug-2021
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6060 - International Standard published
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05-Aug-2021
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TECHNICAL ISO/TS
SPECIFICATION 23034
First edition
2021-08
Nanotechnologies — Method to
estimate cellular uptake of carbon
nanomaterials using optical
absorption
Nanotechnologies — Méthode d'estimation de la captation cellulaire
des nanomatériaux carbonés par mesure d'absorption optique
Reference number
ISO/TS 23034:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 23034:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23034:2021(E)
Contents Page

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

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

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

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

3 Terms, definitions and abbreviated terms ................................................................................................................................ 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Abbreviated terms ............................................................................................................................................................................... 1

4 Method overview .................................................................................................................................................................................................. 2

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

4.2 Optical absorption of carbon nanomaterials ................................................................................................................ 3

4.3 Optical absorption of biomolecules ...................................................................................................................................... 4

4.4 Determination of the concentration of CNMs in dispersion by absorbance ...................................... 4

4.5 Case studies ............................................................................................................................................................................................... 4

5 Materials and apparatus .............................................................................................................................................................................. 5

5.1 Materials ....................................................................................................................................................................................................... 5

5.1.1 Chemicals ............................................................................................................................................................................... 5

5.1.2 Cell line ..................................................................................................................................................................................... 5

5.2 Apparatus .................................................................................................................................................................................................... 5

5.2.1 UV-Vis-NIR spectrometer ......................................................................................................................................... 5

5.2.2 Cuvette for optical absorption measurement ......................................................................................... 5

5.2.3 Incubator, 37 °C, humidified, 5 % CO /air. ................................................................................................ 5

5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well

plates with flat bottom are recommended. ............................................................................................... 5

5.2.5 Centrifuge. ............................................................................................................................................................................. 5

5.2.6 Homogenizer. ...................................................................................................................................................................... 6

5.2.7 Cell counter. .......................................................................................................................................................................... 6

6 Cell-uptake testing protocol ..................................................................................................................................................................... 6

6.1 General ........................................................................................................................................................................................................... 6

6.2 Sample preparation ............................................................................................................................................................................ 6

6.3 Preparation of calibration curve of CNM dispersions ........................................................................................... 6

6.4 Cell-seeding ................................................................................................................................................................................................ 6

6.5 Treatment of cells with testing suspension ................................................................................................................... 7

6.6 Cell counting.............................................................................................................................................................................................. 7

6.7 Washing cells and preparation of the cell lysate ........................................................................................................ 7

6.7.1 General...................................................................................................................................................................................... 7

6.7.2 For adherent cells ............................................................................................................................................................ 7

6.7.3 For floating cells ............................................................................................................................................................... 8

6.8 Absorbance measurement of the cell lysate .................................................................................................................. 8

7 Sources of variability ....................................................................................................................................................................................... 8

8 Data output ................................................................................................................................................................................................................. 9

8.1 General ........................................................................................................................................................................................................... 9

8.2 Data analysis and reporting ......................................................................................................................................................... 9

8.3 Data sheet format ................................................................................................................................................................................. 9

Annex A (informative) Case study with SWCNTs ...................................................................................................................................10

Annex B (informative) Case study of CNHs ..................................................................................................................................................16

Annex C (informative) Case study of MWCNTs .........................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................24

© ISO 2021 – All rights reserved iii
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ISO/TS 23034:2021(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.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 23034:2021(E)
Introduction

Owing to their unusual physical and chemical properties, carbon nanomaterials (CNMs), such as

carbon nanotubes, carbon black, graphene, and carbon nanohorns, have been considered for various

applications such as in the fields of electronics, energy, nanotechnology, and biology. With the increase

of CNM-based products on the market, the public concern regarding possible toxicities has also

increased. Estimation of the amount of CNM associated with the targeted cells is useful for an initial

[1][2][3][4]
toxicological screening of CNMs and for developing applications in medicine .

Fluorescent dyes and/or radioactive isotopes have been routinely used to measure cellular uptake.

Because CNMs absorb light in near infrared (NIR) region, where the bio-components such as protein

and water in cells or tissues have relatively low light absorption, the cellular uptake of CNMs can be

[5][6][7][8]
estimated from the absorbance of cell-lysate .
© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 23034:2021(E)
Nanotechnologies — Method to estimate cellular uptake of
carbon nanomaterials using optical absorption
1 Scope

This document describes a near-infrared optical absorption method to estimate the in vitro cellular

uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell

membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake;

additional analysis using a different technique can be required if quantification is desired.

2 Normative references

The following document is referenced 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 3696:1987, Water for analytical laboratory use — Specification and test methods

ISO/TS 80004-3, Nanotechnologies — Vocabulary — Part 3: Carbon nano-objects
3 Terms, definitions and abbreviated terms

For the purposes of this document, the following terms, definitions and abbreviations as well as the

terms and definitions given in ISO/TS 80004-3 apply.

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

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Terms and definitions
3.1.1
cellular uptake
internalization or association of a substance by a living cell
3.1.2
cell lysis
destruction or dissolution of cells with release of contents
3.1.3
absorbance
measure of the capacity of a substance to absorb light at a specified wavelength
3.2 Abbreviated terms
CNH carbon nanohorn
CNM carbon nanomaterial
CNT carbon nanotube
© ISO 2021 – All rights reserved 1
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ISO/TS 23034:2021(E)
SWCNT single-wall carbon nanotube
MWCNT multiwall carbon nanotube
PBS phosphate-buffered saline
SDBS sodium dodecylbenzene sulfonate
NIR near infrared
UV ultraviolet
Vis visible
4 Method overview
4.1 General

When an optical beam goes through a solution sample, some of the beam is attenuated by the solution,

and the rest is transmitted (see Figure 1). The amount of optical beam attenuated by the solution is

related to the property of solution itself and the thickness of the solution sample.

Key
1 incident light
2 transmitted light
Figure 1 — Optical attenuation by a solution sample

The optical absorbance is directly proportional to the concentration of the dissolved substance in a

solution. When the concentration of solution is expressed as g·l , the relationship between absorbance

and concentration can be written as follows.
A = log (I /I) = k tc (1)
10 0 m
where
A is the optical absorbance of the solution sample;
I is the radiant fluxes of incident;
I is the radiant fluxes of transmitted beams;
−1 −1

k is the wavelength-dependent mass absorptivity coefficient with units of g ·cm ;

t is the thickness of the solution sample;

c is the concentration of a substance dissolved in the solution sample expressed in units of g·l .

2 © ISO 2021 – All rights reserved
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ISO/TS 23034:2021(E)

If A, k and t are known, then the concentration of a substance dissolved in the solution sample can

be obtained. A is obtained by measurement, k is obtained by calibration using the known amount of

the substance dissolved in the solution sample, and t is a fixed value determined by a cuvette optical

pathlength for absorbance measurement.
4.2 Optical absorption of carbon nanomaterials

The optical absorption spectra of carbon nanomaterials in dispersion show a strong absorption peak

in 300 nm to 200 nm (4 eV to 6 eV) that is attributed to the collective excitations of π electron systems

[9] [10]

(π-plasmons). This π-plasmon absorption peak can be also observed in most graphitic compounds.

The peak is superimposed on the featureless background extending to the vis-NIR and IR regions. The

typical spectra of SWCNTs, MWCNTs, CNHs, carbon blacks and graphene are shown in Figure 2. The

broad non-resonant absorbance of MWCNTs, CNHs, carbon black, graphene and/or SWCNTs in vis-NIR

region (e.g. 700 nm to 900 nm) is typical for most carbonaceous nanomaterials in samples.

Key
X wavelength (nm) 3 MWCNTs
Y absorbance (arbitrary units) 4 CNHs
1 individual SWCNTs 5 carbon black
2 SWCNTs bundles 6 graphene

NOTE The oblique regions show the absorbance of each carbon nanomaterial in 700 nm to 900 nm.

Figure 2 — Typical absorption spectra of individual SWCNTs, SWCNT-bundles, MWCNTs, CNHs,

carbon black and graphene nanoplates in dispersions
© ISO 2021 – All rights reserved 3
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ISO/TS 23034:2021(E)
4.3 Optical absorption of biomolecules

Cell lysate may contain the bio-components, such as proteins, amino acids, fatty acids and DNA, which

can absorb light at wavelengths ranged from 200 nm to 600 nm. Water has absorption in IR (> 1 000 nm)

(see Figure 3). Because the absorption of the bio-components in the region of 650 nm to 900 nm is low,

[11]
this region is always used for diagnosis and also called as therapeutic window .
Key
X wavelength (nm)
Y absorbance (arbitrary units)
1 low absorption region
Figure 3 — Absorption spectrum of haemoglobin (Hb) and water
4.4 Determination of the concentration of CNMs in dispersion by absorbance

As shown above, when the light wavelength and the path length of light are fixed, the amount of absorbed

light by a certain CNM dispersion would be directly proportional to the concentration of the dispersed

CNMs. Then based on a calibration curve obtained by known concentration and its corresponding

absorbance, the unknown concentration of CNMs dispersion can be determined from its absorbance.

Since most types of CNMs have absorbance in 700 nm to 900 nm where the components in cell lysis do

not, any wavelength in this region (e.g. 750 nm) can be chosen to determine the concentration of CNMs

dispersion.

NOTE This technique is limited to carbon nanomaterials that absorb strongly in the NIR region of 700 nm

to 900 nm but that would not be suitable for some types of carbon nanomaterials such as nanodiamond and

nanographene oxide, which have low absorbance in this region.
4.5 Case studies

Case studies with SWCNTs, CNHs and MWCNTs are presented in Annexes A, B and C respectively.

4 © ISO 2021 – All rights reserved
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ISO/TS 23034:2021(E)
5 Materials and apparatus
5.1 Materials
5.1.1 Chemicals

5.1.1.1 Water, deionized and sterilized pure water, grade 1, in accordance with ISO 3696:1987.

5.1.1.2 Culture medium, with or without serum that meets the growth requirements of the selected

cell line.
5.1.1.3 PBS (pH = 7,4).

5.1.1.4 Cell lysis reagent, a colourless buffer solution that contains detergents for mammalian cell

lysis/extraction.

5.1.1.5 SDBS solution, SDBS powder dissolved in deionized pure water with concentration of 50 mg/

ml.
5.1.1.6 Trypsin-EDTA (0,25 %).
5.1.2 Cell line

Established cell lines are preferred and shall be obtained from recognized repositories. They can be

either adherent cells or floating cells.
5.2 Apparatus
5.2.1 UV-Vis-NIR spectrometer

A spectrophotometer covering a broad ultraviolet to NIR wavelength range shall be used. Equipment

should be installed in a clean environment, while avoiding any electrical noise, mechanical vibrations,

direct sunlight, etc.

The spectrophotometer should be turned on 30 min prior to the measurement to allow the baseline to

stabilize. The spectrophotometer should be calibrated in the absorbance scale, where an absorptive

filter of neutral optical density with a value close to zero may be used. An absorption spectrum of CNMs

aqueous dispersion should be obtained against a reference of the solution that used for dissolution of

cells [e.g. a mixture solution of SDBS and cell lysis reagent (1:1)].
5.2.2 Cuvette for optical absorption measurement
Quartz cuvette.
5.2.3 Incubator, 37 °C, humidified, 5 % CO /air.

5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well plates with flat

bottom are recommended.
5.2.5 Centrifuge.

Centrifuge tubes: Sterilized, 13-ml centrifuge tubes and 1,5 ml centrifuge micro-tubes.

Centrifuge: Refrigerated centrifuge equipped with rotors of 15-ml centrifuge tubes and 1 ml to 2 ml

micro-tubes. The centrifuge speed in gravitation force is recommended to be 150 g.

© ISO 2021 – All rights reserved 5
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ISO/TS 23034:2021(E)
5.2.6 Homogenizer.
An ultra-sonicator with a horn tip with a minimum output power of 200 W.
For convenience, an ultra-sonicator with multi-tips is recommended.
5.2.7 Cell counter.
Automated cell counter, or hemacytometer.
6 Cell-uptake testing protocol
6.1 General
[12][13]

Follow the basic principle of cell culture techniques regarding expanding a frozen stock of cells so

that the cell uptake for CNM can be performed. The processes described below are recommended when

the 6-well plates are used for cell culture. It can be modified according to the cell-lines and the types of

cell culture dishes.
This method is applicable to all cell types.
6.2 Sample preparation

CNM suspensions for cellular uptake testing should be homogeneous, and stable in aqueous solution. It

can contain dispersant such as bovine serum albumin or polyethylene glycol etc. The concentrations of

CNMs should be known before use. It is suggested to use freshly prepared CNM dispersions.

6.3 Preparation of calibration curve of CNM dispersions

Prepare CNM dispersions with various concentrations such as 0 µg/ml, 0,1 µg/ml, 0,5 µg/ml, 1 µg/ml,

2 µg/ml, 5 µg/ml and 10 µg/ml by dilution of the testing suspension (see 5.1.1) with a solution that used

for dissolving cells [e.g. a mixture solution of SDBS and cell lysis reagent (1:1)]. Collect the absorbance

value for each concentration of CNM dispersion in the cuvette at a consistent wavelength in the region

of 700 nm to 900 nm, for example 750 nm, using the UV-Vis-NIR spectrometer. Prepare the calibration

curve by plotting absorbance at the chosen wavelength against known CNM concentration.

NOTE 1 The calibration curve of the CNM-concentration to absorbance is dependent on the characteristics of

CNMs and their dispersion.

The calibration curve should be prepared for each CNM type used for cellular uptake experiment.

NOTE 2 The absorption of CNMs in 700 nm to 900 nm is the cumulative absorbance of the total of the

carbonaceous material, including impurities such as amorphous carbon, graphite, etc. It is preferable to use

purified CNM samples.
6.4 Cell-seeding
5 5

The cells in culture medium at 2,0 × 10 to 8,0 × 10 in 3 ml culture medium shall be seeded in each

well of 6-well plates and incubated in a humidified incubator at 37 °C with 5 % CO for 24 h. For cellular

uptake measurement, 3-plates of cells, called groups, should be prepared. One plate each for cell

counting, control of cell lysis, and CNM-testing (see Figure 4).
6 © ISO 2021 – All rights reserved
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ISO/TS 23034:2021(E)
a) Control for cell counting b) Control (n = 6) c) CNM testing (n = 6)

NOTE Each plate corresponds to either a control for cell counting, or control for cell lysis, or for CNM-testing.

Figure 4 — Schematic plates for cell culture
6.5 Treatment of cells with testing suspension

Directly add CNM testing suspension or PBS (control) into cell medium and make sure the concentration

of CNM in cell medium is below the one which is toxic to cells (the cell viability is lower than 95 % after

treatment with testing suspension). Gently transfer the plates to incubator and incubate cells for 24 h

[14]

to 48 h. Here, the concentration of CNMs is suggested to be in the range of 10 µg/ml to 100 µg/ml.

However this concentration should be adjusted specific to each cell type and carbon nanomaterial and

to make sure that the cell viability is larger than 95 % of control. The cell viability can be estimated by

using cell proliferation assay reagents such as WST-1 or MTT assay according to the protocol provided

by the manufacturer (the MTS assay in ISO 19007:2018 can be referenced).
6.6 Cell counting
[12][13]

Cell numbers in each well can be verified with a cell counter or haemacytometer. The detail

procedure is described in ISO 20391-1:2018.
6.7 Washing cells and preparation of the cell lysate
6.7.1 General

The washing procedures below are described in detail because they are a significant step in this

protocol. However, the processes described below is recommended when the 6-well dishes are used. It

can be modified according to the cell lines and the types of culture dishes.
6.7.2 For adherent cells

a) After above incubation (see 6.5), gently remove the culture medium with or without CNMs.

b) For washing, gently pipette 2-ml PBS into each well by careful adding solution along the side of the

well to minimize disruption to adherent cells. Gently remove the 2-ml PBS via pipette.

c) Repeat b) three times.

NOTE The steps b) and c) can be modified if necessary for certain cell types by harvesting the cells with

detachment agent such as trypsin and then washing cells by centrifuge with PBS twice.

d) Add 0,5 ml solution of cell lysis reagent into each well.
e) Keep the plates in a laminar flow cabinet at room temperature for 30 min.

f) Mix the solution in the well by gently pipetting several times. Transfer the above solution from each

well one by one to 1,5 ml micro-tubes.

g) Add 0,5 ml SDBS solution into each well and pipetting several times to disperse any materials on

the side or bottom of well into solution.
© ISO 2021 – All rights reserved 7
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ISO/TS 23034:2021(E)

h) Remove the solution from each well and add it to each respective 1,5 ml micro-tubes.

i) Set the 1,5 ml micro-tube in ice water and treat the solution with a homogenizer one by one or

multiply for that homogenizer with one or multi-tips (see 5.2.6).
6.7.3 For floating cells

a) After the incubation (see 6.5), transfer the cells and culture medium with or without CNMs to a 15-

ml centrifuge tube.

b) Wash each well with 1 ml fresh culture medium and transfer into corresponding 15 ml centrifuge

tube.
c) Centrifuge cell suspension at 150 g for 5 min at 4 °C.
d) Remove the supernatant.

e) Add 3-ml PBS into each tube and re-disperse cells by pipetting via a pipette with 1-ml tip.

f) Repeat above process of c) to e) for three times to remove CNMs in outside of cells.

g) Add 0,5 ml solution of cell lysis reagent into each obtained cell-pellet.

h) Disperse the cells by pipetting several times and then keep the tubes in a laminar flow cabinet at

room temperature for 30 min.

i) Transfer the above solution from each tube to 1,5 ml micro-tubes one by one after pipetting several

times.

j) Wash each 15-ml centrifuge tube with 0,5 ml SDBS solution and then transfer them to above 1,5 ml

micro-tubes respectively.

k) Set the 1,5 ml micro-tube in ice water and treat the solution with a homogenizer (see 5.2.6).

6.8 Absorbance measurement of the cell lysate

Measurement wavelength can be set at a wavelength in 700 nm to 900 nm (e.g. 750 nm). The reference

control blank should be measured by using a mixture solution of SDBS and cell lysis reagent (1:1).

Transfer cell lysate [see 6.7.2, i) or 6.7.3, k)] into a spectrophotometer (see 5.2.2) and measure the

absorbance of each sample individually at a wavelength in 700 nm to 900 nm (e.g. 750 nm).

7 Sources of variability

Sources of variability in the results for screening cellular uptake of CNMs by the method proposed in

this document could be traced to, for example:

a) The cellular uptake quantity of CNMs estimated by the method provided in this document contain

CNMs as well as its impurities of non-CNM impurities.

b) If the cell lysates are not dispersed homogeneously, the measurement result might be incorrect due

to the light scatting attenuated light intensity.

c) The number of cells in the CNM testing group might be different from the control group (non-

CNMs addition).
...

TECHNICAL ISO/TS
SPECIFICATION 23034
First edition
Nanotechnologies — Method to
estimate cellular uptake of carbon
nanomaterials using optical
absorption
Nanotechnologies — Méthode d'estimation de la captation cellulaire
des nanomatériaux carbonés par mesure d'absorption optique
PROOF/ÉPREUVE
Reference number
ISO/TS 23034:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 23034:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23034:2021(E)
Contents Page

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

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

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

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

3 Terms, definitions and abbreviated terms ................................................................................................................................ 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Abbreviated terms ............................................................................................................................................................................... 1

4 Method overview .................................................................................................................................................................................................. 2

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

4.2 Optical absorption of carbon nanomaterials ................................................................................................................ 3

4.3 Optical absorption of biomolecules ...................................................................................................................................... 4

4.4 Determination of the concentration of CNMs in dispersion by absorbance ...................................... 4

4.5 Case studies ............................................................................................................................................................................................... 4

5 Materials and apparatus .............................................................................................................................................................................. 5

5.1 Materials ....................................................................................................................................................................................................... 5

5.1.1 Chemicals ............................................................................................................................................................................... 5

5.1.2 Cell line ..................................................................................................................................................................................... 5

5.2 Apparatus .................................................................................................................................................................................................... 5

5.2.1 UV-Vis-NIR spectrometer ......................................................................................................................................... 5

5.2.2 Cuvette for optical absorption measurement ......................................................................................... 5

5.2.3 Incubator, 37 °C, humidified, 5 % CO /air. ................................................................................................ 5

5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well

plates with flat bottom are recommended. ............................................................................................... 5

5.2.5 Centrifuge. ............................................................................................................................................................................. 5

5.2.6 Homogenizer. ...................................................................................................................................................................... 6

5.2.7 Cell counter. .......................................................................................................................................................................... 6

6 Cell-uptake testing protocol ..................................................................................................................................................................... 6

6.1 General ........................................................................................................................................................................................................... 6

6.2 Sample preparation ............................................................................................................................................................................ 6

6.3 Preparation of calibration curve of CNM dispersions ........................................................................................... 6

6.4 Cell-seeding ................................................................................................................................................................................................ 6

6.5 Treatment of cells with testing suspension ................................................................................................................... 7

6.6 Cell counting.............................................................................................................................................................................................. 7

6.7 Washing cells and preparation of the cell lysate ........................................................................................................ 7

6.7.1 General...................................................................................................................................................................................... 7

6.7.2 For adherent cells ............................................................................................................................................................ 7

6.7.3 For floating cells ............................................................................................................................................................... 8

6.8 Absorbance measurement of the cell lysate .................................................................................................................. 8

7 Sources of variability ....................................................................................................................................................................................... 8

8 Data output ................................................................................................................................................................................................................. 9

8.1 General ........................................................................................................................................................................................................... 9

8.2 Data analysis and reporting ......................................................................................................................................................... 9

8.3 Data sheet format ................................................................................................................................................................................. 9

Annex A (informative) Case study with SWCNTs ...................................................................................................................................10

Annex B (informative) Case study of CNHs ..................................................................................................................................................16

Annex C (informative) Case study of MWCNTs .........................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................24

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 23034:2021(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

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This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

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ISO/TS 23034:2021(E)
Introduction

Owing to their unusual physical and chemical properties, carbon nanomaterials (CNMs), such as

carbon nanotubes, carbon black, graphene, and carbon nanohorns, have been considered for various

applications such as in the fields of electronics, energy, nanotechnology, and biology. With the increase

of CNM-based products on the market, the public concern regarding possible toxicities has also

increased. Estimation of the amount of CNM associated with the targeted cells is useful for an initial

[3][4][5][6]
toxicological screening of CNMs and for developing applications in medicine .

Fluorescent dyes and/or radioactive isotopes have been routinely used to measure cellular uptake.

Because CNMs absorb light in near infrared (NIR) region, where the bio-components such as protein

and water in cells or tissues have relatively low light absorption, the cellular uptake of CNMs can be

[7][8][9][10]
estimated from the absorbance of cell-lysate .
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TECHNICAL SPECIFICATION ISO/TS 23034:2021(E)
Nanotechnologies — Method to estimate cellular uptake of
carbon nanomaterials using optical absorption
1 Scope

This document describes a near-infrared optical absorption method to estimate the in vitro cellular

uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell

membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake;

additional analysis using a different technique can be required if quantification is desired.

2 Normative references

The following document is referenced 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 3696:1987, Water for analytical laboratory use — Specification and test methods

ISO/TS 80004-3, Nanotechnologies — Vocabulary — Part 3: Carbon nano-objects
3 Terms, definitions and abbreviated terms

For the purposes of this document, the following terms, definitions and abbreviations as well as the

terms and definitions given in ISO/TS 80004-3 apply.

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

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Terms and definitions
3.1.1
cellular uptake
internalization or association of a substance by a living cell
3.1.2
cell lysis
destruction or dissolution of cells with release of contents
3.1.3
absorbance
measure of the capacity of a substance to absorb light at a specified wavelength
3.2 Abbreviated terms
CNH carbon nanohorn
CNM carbon nanomaterial
CNT carbon nanotube
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SWCNT single-wall carbon nanotube
MWCNT multiwall carbon nanotube
PBS phosphate-buffered saline
SDBS sodium dodecylbenzene sulfonate
NIR near infrared
UV ultraviolet
Vis visible
4 Method overview
4.1 General

When an optical beam goes through a solution sample, some of the beam is attenuated by the solution,

and the rest is transmitted (see Figure 1). The amount of optical beam attenuated by the solution is

related to the property of solution itself and the thickness of the solution sample.

Key
1 incident light
2 transmitted light
Figure 1 — Optical attenuation by a solution sample

The optical absorbance is directly proportional to the concentration of the dissolved substance in a

solution. When the concentration of solution is expressed as g·L , the relationship between absorbance

and concentration can be written as follows.
A = log (I /I) = k tc (1)
10 0 m
where
A is the optical absorbance of the solution sample;
I is the radiant fluxes of incident;
I is the radiant fluxes of transmitted beams;
−1 −1

k is the wavelength-dependent mass absorptivity coefficient with units of g ·cm ;

t is the thickness of the solution sample;

c is the concentration of a substance dissolved in the solution sample expressed in units of g·l .

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ISO/TS 23034:2021(E)

If A, k and t are known, then the concentration of a substance dissolved in the solution sample can

be obtained. A is obtained by measurement, k is obtained by calibration using the known amount of

the substance dissolved in the solution sample, and t is a fixed value determined by a cuvette optical

pathlength for absorbance measurement.
4.2 Optical absorption of carbon nanomaterials

The optical absorption spectra of carbon nanomaterials in dispersion show a strong absorption peak

in 300 nm to 200 nm (4 eV to 6 eV) that is attributed to the collective excitations of π electron systems

[1] [11]

(π-plasmons). This π-plasmon absorption peak can be also observed in most graphitic compounds.

The peak is superimposed on the featureless background extending to the vis-NIR and IR regions. The

typical spectra of SWCNTs, MWCNTs, CNHs, carbon blacks and graphene are shown in Figure 2. The

broad non-resonant absorbance of MWCNTs, CNHs, carbon black, graphene and/or SWCNTs in vis-NIR

region (e.g. 700 nm to 900 nm) is typical for most carbonaceous nanomaterials in samples.

Key
X wavelength (nm) 3 MWCNTs
Y absorbance (arbitrary units) 4 CNHs
1 individual SWCNTs 5 carbon black
2 SWCNTs bundles 6 graphene

NOTE The oblique regions show the absorbance of each carbon nanomaterial in 700 nm to 900 nm.

Figure 2 — Typical absorption spectra of individual SWCNTs, SWCNT-bundles, MWCNTs, CNHs,

carbon black and graphene nanoplates in dispersions
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4.3 Optical absorption of biomolecules

Cell lysate may contain the bio-components, such as proteins, amino acids, fatty acids and DNA, which

can absorb light at wavelengths ranged from 200 nm to 600 nm. Water has absorption in IR (> 1 000 nm)

(Figure 3). Because the absorption of the bio-components in the region of 650 nm to 900 nm is low, this

[12]
region is always used for diagnosis and also called as therapeutic window .
Key
X wavelength (nm)
Y absorbance (arbitrary units)
1 no absorption
Figure 3 — Absorption spectrum of haemoglobin (Hb) and water
4.4 Determination of the concentration of CNMs in dispersion by absorbance

As shown above, when the light wavelength and the path length of light are fixed, the amount of absorbed

light by a certain CNM dispersion would be directly proportional to the concentration of the dispersed

CNMs. Then based on a calibration curve obtained by known concentration and its corresponding

absorbance, the unknown concentration of CNMs dispersion can be determined from its absorbance.

Since most types of CNMs have absorbance in 700 nm to 900 nm where the components in cell lysis do

not, any wavelength in this region (e.g. 750 nm) can be chosen to determine the concentration of CNMs

dispersion.

NOTE This technique is limited to carbon nanomaterials that absorb strongly in the NIR region of 700 nm

to 900 nm, but would not be suitable for some types of carbon nanomaterials such as nanodiamond and

nanographene oxide, which have low absorbance in this region.
4.5 Case studies

Case studies with single-wall carbon nanotubes SWCNTs, CNHs and multiwall carbon nanotubes

(MWCNTs) are presented in Annexes A, B and C respectively.
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5 Materials and apparatus
5.1 Materials
5.1.1 Chemicals

5.1.1.1 Water, deionized and sterilized pure water, grade 1, in accordance with ISO 3696:1987.

5.1.1.2 Culture medium, with or without serum that meets the growth requirements of the selected

cell line.
5.1.1.3 Phosphate-Buffered Saline, (PBS; pH = 7,4).

5.1.1.4 Cell lysis reagent, a colourless buffer solution that contains detergents for mammalian cell

lysis/extraction.

5.1.1.5 SDBS solution, Sodium dodecylbenzene sulfonate (SDBS) powder dissolved in deionized pure

water with concentration of 50 mg/ml.
5.1.1.6 Trypsin-EDTA (0,25 %).
5.1.2 Cell line

Established cell lines are preferred and shall be obtained from recognized repositories. They can be

either adherent cells or floating cells.
5.2 Apparatus
5.2.1 UV-Vis-NIR spectrometer

A spectrophotometer covering a broad ultraviolet to NIR wavelength range shall be used. Equipment

should be installed in a clean environment, while avoiding any electrical noise, mechanical vibrations,

direct sunlight, etc.

The spectrophotometer should be turned on 30 min prior to the measurement to allow the baseline to

stabilize. The spectrophotometer should be calibrated in the absorbance scale, where an absorptive

filter of neutral optical density with a value close to zero may be used. An absorption spectrum of CNMs

aqueous dispersion should be obtained against a reference of the solution that used for dissolution of

cells [e.g. a mixture solution of SDBS and cell lysis reagent (1:1)].
5.2.2 Cuvette for optical absorption measurement
Quartz cuvette.
5.2.3 Incubator, 37 °C, humidified, 5 % CO /air.

5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well plates with flat

bottom are recommended.
5.2.5 Centrifuge.

Centrifuge tubes: Sterilized, 13-ml centrifuge tubes and 1,5 ml centrifuge micro-tubes.

Centrifuge: Refrigerated centrifuge equipped with rotors of 15-ml centrifuge tubes and 1 ml to 2 ml

micro-tubes. The centrifuge speed in gravitation force is recommended to be 150 g.

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5.2.6 Homogenizer.
An ultra-sonicator with a horn tip with a minimum output power of 200 W.
For convenience, an ultra-sonicator with multi-tips is recommended.
5.2.7 Cell counter.
Automated cell counter, or hemacytometer.
6 Cell-uptake testing protocol
6.1 General
[13][14]

Follow the basic principle of cell culture techniques regarding expanding a frozen stock of cells so

that the cell uptake for CNM can be performed. The processes described below are recommended when

the 6-well plates are used for cell culture. It can be modified according to the cell-lines and the types of

cell culture dishes.
This method is applicable to all cell types.
6.2 Sample preparation

CNM suspensions for cellular uptake testing should be homogeneous, and stable in aqueous solution. It

can contain dispersant such as bovine serum albumin or polyethylene glycol etc. The concentrations of

CNMs should be known before use. It is suggested to use freshly prepared CNM dispersions.

6.3 Preparation of calibration curve of CNM dispersions

Prepare CNM dispersions with various concentrations such as 0, 0,1 µg/ml, 0,5 µg/ml, 1 µg/ml, 2 µg/

ml, 5 µg/ml and 10 µg/ml by dilution of the testing suspension (see 5.1.1) with a solution that used

for dissolving cells [e.g. a mixture solution of SDBS and cell lysis reagent (1:1)]. Collect the absorbance

value for each concentration of CNM dispersion in the cuvette at a consistent wavelength in the region

of 700 nm to 900 nm, for example 750 nm, using the UV-Vis-NIR spectrometer. Prepare the calibration

curve by plotting absorbance at the chosen wavelength against known CNM concentration.

NOTE 1 The calibration curve of the CNM-concentration to absorbance is dependent on the characteristics of

CNMs and their dispersion.

The calibration curve should be prepared for each CNM type used for cellular uptake experiment.

NOTE 2 The absorption of CNMs in 700 nm to 900 nm is the cumulative absorbance of the total of the

carbonaceous material, including impurities such as amorphous carbon, graphite, etc. It is preferable to use

purified CNM samples.
6.4 Cell-seeding
5 5

The cells in culture medium at 2,0 × 10 to 8,0 × 10 in 3 ml culture medium shall be seeded in each

well of 6-well plates and incubated in a humidified incubator at 37 °C with 5 % CO for 24 h. For cellular

uptake measurement, 3-plates of cells, called groups, should be prepared. One plate each for cell

counting, control of cell lysis, and CNM-testing (see Figure 4).
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a) Control for cell counting
b) Control (n = 6) c) CNM testing (n = 6)

NOTE Each plate corresponds to either a control for cell counting, or control for cell lysis, or for CNM-testing.

Figure 4 — Schematic plates for cell culture
6.5 Treatment of cells with testing suspension

Directly add CNM testing suspension or PBS (control) into cell medium and make sure the concentration

of CNM in cell medium is below the one which is toxic to cells (the cell viability is lower than 95 % after

treatment with testing suspension). Gently transfer the plates to incubator and incubate cells for 24 h

[15]

to 48 h. Here, the concentration of CNMs is suggested to be in the range of 10 µg/ml to 100 µg/ml.

However this concentration should be adjusted specific to each cell type and carbon nanomaterial and

to make sure that the cell viability is larger than 95 % of control. The cell viability can be estimated by

using cell proliferation assay reagents such as WST-1 or MTT assay according to the protocol provided

by the manufacturer (the MTS Assay in ISO 19007:2018 can be referenced).
6.6 Cell counting
[13][14]

Cell numbers in each well can be verified with a cell counter or hemacytometer. The detail

procedure is described in ISO 20391-1:2018.
6.7 Washing cells and preparation of the cell lysate
6.7.1 General

The washing procedures below are described in detail because they are a significant step in this

protocol. However, the processes described below is recommended when the 6-well dishes are used. It

can be modified according to the cell lines and the types of culture dishes.
6.7.2 For adherent cells

a) After above incubation (see 6.5), gently remove the culture medium with or without CNMs.

b) For washing, gently pipette 2-ml PBS into each well by careful adding solution along the side of the

well to minimize disruption to adherent cells. Gently remove the 2-ml PBS via pipette.

c) Repeat b) three times.

NOTE The steps b) and c) can be modified if necessary for certain cell types by harvesting the cells with

detachment agent such as trypsin and then washing cells by centrifuge with PBS twice.

d) Add 0,5 ml solution of cell lysis reagent into each well.
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e) Keep the plates in a laminar flow cabinet at room temperature for 30 min.

f) Mix the solution in the well by gently pipetting several times. Transfer the above solution from each

well one by one to 1,5 ml micro-tubes.

g) Add 0,5 ml SDBS solution into each well and pipetting several times to disperse any materials on

the side or bottom of well into solution.

h) Remove the solution from each well and add it to each respective 1,5 ml micro-tubes.

i) Set the 1,5 ml micro-tube in ice water and treat the solution with a homogenizer one by one or

multiply for that homogenizer with one or multi-tips (see 5.2.6).
6.7.3 For floating cells

a) After the incubation (see 6.5), transfer the cells and culture medium with or without CNMs to a 15-

ml centrifuge tube.

b) Wash each well with 1 ml fresh culture medium and transfer into corresponding 15 ml centrifuge

tube.
c) Centrifuge cell suspension at 150 g for 5 min at 4 °C.
d) Remove the supernatant.

e) Add 3-ml PBS into each tube and re-disperse cells by pipetting via a pipette with 1-ml tip.

f) Repeat above process of c) to e) for three times to remove CNMs in outside of cells.

g) Add 0,5 ml solution of cell lysis reagent into each obtained cell-pellet.

h) Disperse the cells by pipetting several times and then keep the tubes in a laminar flow cabinet at

room temperature for 30 min.

i) Transfer the above solution from each tube to 1,5 ml micro-tubes one by one after pipetting several

times.

j) Wash each 15-ml centrifuge tube with 0,5 ml SDBS solution and then transfer them to above 1,5 ml

micro-tubes respectively.

k) Set the 1,5 ml micro-tube in ice water and treat the solution with a homogenizer (see 5.2.6).

6.8 Absorbance measurement of the cell lysate

Measurement wavelength can be set at a wavelength in 700 nm to 900 nm (e.g. 750 nm). The reference

control blank should be measured by using a mixture solution of SDBS and cell lysis reagent (1:1).

Transfer cell lysate [see 6.7.2, i) or 6.7.3, k)] into a spectrophotometer (see 5.2.2) and measure the

absorbance of each sample individually at a wavelength in 700 nm to 900 nm (e.g. 750 nm).

7 Sources of variability

Sources of variability in the results for screening cellular uptake of CNMs by the method proposed in

this document could be traced to, for example:

a) The cellular uptake quantity of CNMs estimated by the method provided in this document contain

CNMs as well as its impurities of non-CNM impurities.
b) If the cell l
...

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