ISO/TS 7833:2024

Nanotechnologies - Extraction method of nanomaterials from lung tissue by proteinase K digestion

This document provides an extraction method using the proteinase K (PK) for nanomaterials deposited in the lung. This document specifies the advantages of the PK digestion method and examples of nanomaterials to which it can be applied. This document focuses on extracting nanomaterials from lung tissue and separating nanoparticles from their ionic counterparts. This method is potentially (or theoretically) applicable to any particles that are insoluble during the PK digestion process.

Nanotechnologies — Méthode d'extraction de nanomatériaux d'organes par digestion par protéinase K

General Information

• Status: Published
• Publication Date: 30-Jan-2024
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229 - Nanotechnologies
• Current Stage: 6060 - International Standard published
• Start Date: 31-Jan-2024
• Due Date: 09-Aug-2024
• Completion Date: 31-Jan-2024

Overview

ISO/TS 7833:2024 - Nanotechnologies - Extraction method of nanomaterials from lung tissue by proteinase K digestion - specifies an enzymatic extraction method using proteinase K (PK) to recover nanomaterials deposited in lung tissue. The Technical Specification describes the advantages of PK digestion over aggressive chemical digestion (reduced particle dissolution and structural damage) and focuses on separating particulate nanomaterials from their ionic counterparts for subsequent instrumental analysis.

Key topics and technical requirements

• Scope: Extraction of nanomaterials from lung tissue and separation of nanoparticles from dissolved ionic species; method is applicable to particles that remain insoluble during PK digestion.
• Optimal PK system:
  • Recommended digestion buffer (tested formulation): 30 mM Tris‑HCl, 10 mM EDTA, 1% SDS, 5 mM CaCl2, pH 8.0.
  • Optimal PK activity reported in the document: 10 µg PK (≈ 0.2–0.3 U) under the described test conditions.
  • Incubation condition example: 56 °C ± 1 °C (heat block or water bath), typical digestion times in tests were 24 h.
• Sample preparation & equipment:
  • Drying/weighting of lung tissue, slicing (~2 mm), homogenisation (micro ball mill), optional lyophiliser.
  • Vessels resistant to protein adsorption (1.5–50 ml tubes), Petri dishes for drying.
  • Centrifugation capable of ≥21 000 g (microcentrifuge/ultracentrifuge) to pellet nanoparticles; bath sonicator (≈400 W, 40 kHz) for dispersion.
• Procedure outline:
  • Sampling and drying, homogenisation/powderisation, PK digestion of tissue, collection of nanoparticle pellet and preparation for instrumental analysis.
• Methodological considerations:
  • Guidelines on separating particulate and ionic fractions, nanomaterials types applicable (e.g., carbon-based materials and certain metal oxides), and the impact of blood content or organ-specific variability.

Applications and users

• Primary applications:
  • Lung burden analysis in inhalation toxicology and toxicokinetic studies.
  • Recovery and characterization of retained nanomaterials prior to ICP‑MS, sp-ICP‑MS, TEM, EDS, UV‑Vis or other instrumental analyses.
• Who should use this standard:
  • Nanotoxicology laboratories, regulatory testing labs, contract research organisations (CROs), materials scientists, and researchers conducting inhalation or organ-distribution studies of engineered nanomaterials.

Related standards and guidance

• ISO 80004‑1 (Nanotechnologies - Vocabulary)
• ISO/TR 22019 (toxicokinetic considerations for nanomaterials)
• OECD inhalation guidance (TG 412, TG 413) and ongoing toxicokinetic guidance initiatives (related to TG 417 discussions)

ISO/TS 7833:2024 provides a practical, validated PK-based protocol to extract particulate nanomaterials from lung tissue while minimising artefacts from chemical digestion - a key tool for accurate lung burden and toxicokinetic assessments.

Standards Content (Sample)

Technical
Specification
ISO/TS 7833
First edition
Nanotechnologies — Extraction
2024-01
method of nanomaterials from lung
tissue by proteinase K digestion
Nanotechnologies — Méthode d'extraction de nanomatériaux
d'organes par digestion par protéinase K
Reference number
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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or ISO’s member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms. 2
5 Materials - PK digestion buffer and optimal concentration for lung tissue digestion . 2
6 Technical equipment . 2
6.1 Vessels .2
6.2 Heat block or water bath .3
6.3 Drying oven .3
6.4 Micro ball mill .3
6.5 Microcentrifuge or ultracentrifuge .3
6.6 Bath sonicator .3
6.7 Pipettes .3
7 Procedures . 3
7.1 Preparation of lung tissue sample for digestion .3
7.1.1 Sampling and drying for lung tissue samples .3
7.1.2 Homogenisation of dried tissue slices .3
7.2 Tissue digestion by PK .3
7.3 Collection of nanomaterials and preparation for instrumental analysis .4
8 Methodological considerations for the digestion by PK . 4
8.1 Separative collection of nanomaterials from their ionic counterparts .4
8.2 Types of nanomaterials applicable to this method .4
8.3 The impact of blood in organs on this method .4
Annex A (informative) Recovery efficiency of nanomaterials from the spiking experiment and
identification of nanomaterials with TEM after PK digestion . 5
Annex B (informative) Evaluation of the optimal PK digestion buffer for lung tissue digestion . 8
Annex C (informative) Evaluation of the optimal concentration of PK for tissue digestion . 9
Annex D (informative) Efficacy of tissue digestion with/without drying and powderisation .10
Annex E (informative) Comparison of tissue digestion with/without perfusion .11
Bibliography .13

iii
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
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
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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
Introduction
Quantification of nanomaterials deposited in organs is important to evaluate the lung burden in an
[3][4][5]
inhalation toxicity study and organ distribution in toxicokinetics studies . Owing to the long retention
period of nanomaterials deposited in the alveoli in an inhalation setting, the OECD revised the subacute
and subchronic inhalation test guidelines, TG 412 and TG 413 respectively, to include lung burden analysis
[3][4]
when testing poorly soluble nanomaterials . However, the lung burden analysis method varies depending
on the nanomaterials, thus the development of standard methods is highly needed. In addition, a new or
[6][7]
revised OECD toxicokinetics test guideline (i.e. TG417) is needed to accommodate nanomaterials .
Furthermore, OECD launched a new project (1.10) for developing a guidance document on the determination
[8]
of concentrations of nanoparticles in biological samples for (eco)toxicity studies . ISO/TR 22019 addresses
considerations for performing toxicokinetic studies with nanomaterials. However, standard methods to
measure concentrations of nanomaterials deposited in organs are needed to complement TG 412, TG 413
and ISO/TR 22019.
Quantification of nanomaterials in organs can be divided into two steps:
a) collection of nanomaterials from organs;
b) quantification of nanomaterials using instrumental analysis.
To collect nanomaterials deposited in organs, chemical or enzymatic digestion methods can be used. The
ultimate goal of step a) is to collect the particle in particle-form (i.e. the same material that animals were
exposed to) rather than the ionic counterparts. However, many of the chemicals used for digestion such
as hydrogen chloride, nitric acid, and hydrofluoric acid can ionize some nanomaterials or damage their
[9][10]
structure . Among nanomaterials, metals or metal oxides can be dissolved by chemicals for digesting
organs. Thus, the measured amount of metal ions in organs treated with these digestion chemicals would
not be the amount of nanomaterials inhaled. It could be the ionic counterparts of these nanomaterials as well
[11][12]
as the same metal present as endogenous ions in the organ . Although carbon-based nanomaterials
such as carbon nanotubes (CNTs), graphene, and nanodiamonds are not dissolved by chemical digestion,
[9]
the structure of the carbon-based nanomaterials can undergo alterations including defects and oxidation .
The second step is the quantification of nanomaterials by instrumental analysis including methods such as
inductively coupled plasma mass spectrometry (ICP-MS), fluorometry, and optical absorbance spectrometry.
Because the instrumental analysis is diverse and needs to correspond to the physicochemical properties of
the nanomaterial analysed, this document focuses on the method of extracting nanomaterials from organs.
In contrast, the enzymatic digestion of the mixture of powderised lung tissue and nanomaterials in vitro,
and lung tissue instilled nanomaterials in vivo using proteinase K (PK) can successfully dissolve tissues
with less alterations of the structure of carbon-based nanomaterials and many metal oxides compared to the
[13]
chemical digestion method . This method allows to collect nanomaterial particles separately from their
ionic counterparts dissolved in supernatants. In a previous study, the PK digestion successfully digested
lung tissues, and it was possible to separately collect carbon-based nanomaterials including carbon black,
[13]
carbon nanotube, carbon nanofibre, graphene, and nanodiamond . Other studies have also demonstrated
the use of this method to successfully collect and quantify single-walled carbon nanotubes (SWCNTs)
instilled into mouse lung, CNTs spiked into rat lung tissue, and microplastics in marine invertebrates
[9][14][15]
species . However, misleading or inaccurate results may occur if nanomaterials are dissociated
or dissolved during the process of enzymatic digestion. The examples of nanomaterials for which the PK
digestion method is applicable or not applicable are listed in Annex A. Although this document focuses on
the lung tissue digestion, it can be further applicable to other tissues. However, organs besides the lung
should be tested for their validity based on this document because the efficacy of PK for tissue digestion
varies by the organ-specific nature. Therefore, an optimized procedure to extract nanomaterials from lung
tissue is highly needed as a part of recommendations and guidelines on how to conduct lung burden analysis
or toxicokinetic studies.
v
Technical Specification ISO/TS 7833:2024(en)
Nanotechnologies — Extraction method of nanomaterials
from lung tissue by proteinase K digestion
1 Scope
This document provides an extraction method using the proteinase K (PK) for nanomaterials deposited in the
lung. This document specifies the advantages of the PK digestion method and examples of nanomaterials to
which it can be applied. This document focuses on extracting nanomaterials from lung tissue and separating
nanoparticles from their ionic counterparts. This method is potentially (or theoretically) applicable to any
particles that are insoluble during the PK digestion process.
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 80004-1, Nanotechnologies — Vocabulary — Part 1: Core vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in and ISO 80004-1 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
nanoparticle
nano-object with all external dimensions in the nanoscale
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as nanofiber or
nanoplate are preferred to the term nanoparticle.
[SOURCE: ISO 80004-1:2023, 3.3.4]
3.2
nanotube
hollow nanofibre
[SOURCE: ISO 80004-1:2023, 3.3.8]

4 Symbols and abbreviated terms
PK proteinase K
ICP-MS inductively coupled plasma mass spectrometry
ICP-OES inductively coupled plasma optical emission spectroscopy
sp-ICP-MS single particle inductively coupled plasma mass spectrometry
TEM transmission electron microscopy
EDS energy-dispersive X-ray spectroscopy
DMSO dimethyl sulfoxide
CNT carbon nanotube
SWCNT single-walled carbon nanotube
MWCNT multi-walled carbon nanotube
CB carbon black
ND nanodiamond
rGO reduced graphene oxide
UV-Vis Ultraviolet–visible
PBS phosphate-buffered saline
ALF artificial lysosomal fluid
SDS sodium dodecyl sulfate
5 Materials - PK digestion buffer and optimal concentration for lung tissue digestion
2+
Because PK requires activators such as Ca , the addition of CaCl in the digestion buffer act as an activator
[16] [16]
of PK . The protein denaturing agents such as SDS and urea can stimulate the activity of PK . To
select an optimal recipe for PK digestion buffer, four recipes were tested by incubating at 56 °C for 24 h of
homogenised lung tissue with PK at 10 μg (equivalent to 0,2 U to 0,3 U) per milligram dry mass of lung tissue
homogenates (see Annex B). Then, the absorbance of digested samples was tested at 750 nm wavelengths.
From this experiment, an optimal buffer recipe was selected as 30 mM Tris-HCl, 10 mM EDTA, 1 % SDS, 5 mM
CaCl , and pH 8,0 (see Annex B). Then, with the selected PK digestion buffer, the optimal concentration of PK
for lung tissue digestion was selected by incubating various concentrations of PK with the 20 mg dry mass of
lung tissue homogenates (see Annex C). The result showed that the optimal concentration was 10 μg, which
is equivalent to about 0,2 U to 0,3 U. One unit of enzyme liberates Folin-positive amino acids and peptides,
[17]
corresponding to 1 μmol in 1 min at 37 °C using denatured hemoglobin as substrate .
6 Technical equipment
6.1 Vessels
A sterile microcentrifuge tube (1,5 ml or 2 ml) can be used. All tubes and glassware are required to be
resistant to protein adsorption. Based on the amount of tissue, 15 ml or 50 ml tube can be used. A Petri dish
(90 mm × 15 mm) can be used for drying tissue slices.

6.2 Heat block or water bath
Heat block or water bath with controlled temperature (56 °C ± 1 °C) can be used.
6.3 Drying oven
Any drying oven that controls 60 °C can be used. In addition, a lyophilizer that can dry sliced tissue is
optional technical equipment.
6.4 Micro ball mill
The micro ball mill can be used to homogenise (or powderise) tissues.
6.5 Microcentrifuge or ultracentrifuge
Microcentrifuge which can centrifuge at 21 000 g or higher is recommended. Because the speed and
duration of centrifugation are related to physicochemical characteristics of the nanomaterials such as size
and density, an optimal speed and duration should be determined based on whether it can completely spin
down nanomaterials. For nanomaterials that cannot be pelleted by conventional microcentrifuge, a higher
speed centrifuge such as ultracentrifuge can be used.
6.6 Bath sonicator
The bath sonicator can be used. The power of the bath sonicator can vary but normally, 400 W and 40 kHz
are acceptable.
6.7 Pipettes
The single-channel pipette can be used.
7 Procedures
7.1 Preparation of lung tissue sample for digestion
7.1.1 Sampling and drying for lung tissue samples
The collected lung tissue should be weighed before and after drying to report the concentration of
nanomaterials per wet or dry mass of lung tissue. Lung tissue should be sliced into pieces with a diameter of
about 2 mm. The size of tissue slices can vary. The sliced tissues places in a Petri dish and dried at 60 °C for
2 d in
...