ISO/TR 23652:2024

Nanotechnologies — Considerations for radioisotope labelling methods of nanomaterials for performance evaluation

This document provides: a) a review of radioisotope labelling methods that can be used for nanomaterials; b) the advantages and disadvantages of each radioisotope labelling method; c) information on the selection of a matched pair of nanomaterial and radioisotope labelling method to ensure the in vivo integrity of radioisotope-labelled nanomaterials or the stability of their performance.

Nanotechnologies — Considérations relatives aux méthodes de marquage radio-isotopique des nanomatériaux pour l'évaluation des performances

General Information

• Status: Published
• Publication Date: 10-Jun-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: 11-Jun-2024
• Completion Date: 11-Jun-2024

Standards Content (Sample)

Technical
Report
ISO/TR 23652
First edition
Nanotechnologies — Considerations
2024-06
for radioisotope labelling methods
of nanomaterials for performance
evaluation
Nanotechnologies — Considérations relatives aux méthodes de
marquage radio-isotopique des nanomatériaux pour l'évaluation
des performances
Reference number
© ISO 2024
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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 Abbreviated terms . 2
5 Biodistribution study and radioisotopes. 3
5.1 Biodistribution study .3
5.2 Radioisotopes .3
6 Radioisotope labelling methods for nanomaterials . 4
6.1 General .4
6.2 Pre- and post-surface labelling method .6
6.3 Chelating agent-based labelling .6
6.4 Chelating agent-free radioisotope labelling method .8
6.4.1 General .8
6.4.2 Radioactive-plus-non-radioactive precursors .8
6.4.3 Specific trapping .8
6.4.4 Cation exchange .9
6.4.5 Neutron or proton beam activation.10
6.5 Dual radioisotope labelling .10
6.6 Choice of radioisotopes .11
6.7 Production of radioisotopes .11
6.7.1 General .11
6.7.2 Cyclotron-produced radioisotopes . 12
6.7.3 Reactor-produced radioisotopes. 12
6.7.4 Generator-produced radioisotopes . 13
6.8 Chelating agent and the matched pair for radioisotope . 13
7 The stability of radioisotope-labelled nanomaterials . 14
8 Advantages and disadvantages of radioisotope labelling method .15
Annex A (informative) Representative radioisotopes used for nanomaterial labelling .16
Annex B (informative) Advantages and disadvantages of radioisotope labelling methods for
nanomaterials . 17
Bibliography .18

iii
Foreword
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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)
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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
Prior to the clinical trials of nanomaterials intended for use in human medicine, their in vivo behaviour
has been evaluated in animal experiments. Several quantitative methods for assessing the biodistribution
of nanomaterials have been developed. Among these methods, the biodistribution of radioisotope-labelled
nanomaterials provides quantitative information on their distribution throughout the entire body.
The use of radioisotope-labelled nanomaterials for biodistribution studies is a well-established method for
understanding the pharmacokinetics or toxicokinetics of nanomaterials in vivo. These methods assume that
the distribution pattern of nanomaterials and radioisotope-labelled nanomaterials will be similar or nearly
identical in vivo.
Radioisotope labelling of nanomaterials can be accomplished using a wide variety of radionuclides and
associated labelling methods. However, for nanomaterials used for medicinal purposes, there are only a
few matching pairs of nanomaterial and radioisotope labelling method that ensure the in vivo integrity of
the radioisotope-labelled nanomaterial. Failure to identify and apply matching pairs of nanomaterial and
radioisotope labelling method in studies preceding the clinical trial phase can lead to experimental data on
biodistribution in which the nanomaterial and radio-label separate during the experiment. This in turn can
result in a large number of nanomaterials or nano-drugs failing in the clinical trial phase.

v
Technical Report ISO/TR 23652:2024(en)
Nanotechnologies — Considerations for radioisotope
labelling methods of nanomaterials for performance
evaluation
1 Scope
This document provides:
a) a review of radioisotope labelling methods that can be used for nanomaterials;
b) the advantages and disadvantages of each radioisotope labelling method;
c) information on the selection of a matched pair of nanomaterial and radioisotope labelling method
[1]
to ensure the in vivo integrity of radioisotope-labelled nanomaterials or the stability of their
performance.
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
ISO/TS 80004-8, Nanotechnologies — Vocabulary — Part 8: Nanomanufacturing processes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 80004-1, ISO/TS 80004-8 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
nanoscale
length range approximately from 1 nm to 100 nm
[SOURCE: ISO 80004-1:2023, 3.1.1]
3.2
nanomaterial
material with any external dimension in the nanoscale (3.1) or having internal structure or surface structure
in the nanoscale
[SOURCE: ISO 80004-1:2023, 3.1.4, modified — Notes have been removed.]

3.3
nanoparticle
nano-object with all external dimensions in the nanoscale (3.1)
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as nanofibre or
nanoplate are preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-1:2023, 3.3.4]
3.4
radioisotope
unstable isotope of an element that decays or disintegrates spontaneously, emitting ionizing radiation that
can be alpha particles, beta particles and/or gamma rays
Note 1 to entry: Approximately 5 000 natural and artificial radioisotopes have been identified.
[SOURCE: ISO 19461-1:2018, 3.9, modified — "that can be alpha particles, beta particles and/or gamma rays"
has been added to the definition.]
3.5
biodistribution
technique used to monitor the movement and distribution of specific radiolabelled nanomaterials (3.2)
within an experimental animal or human subject
3.6
chelating agent
substance having a molecular structure embodying several electron-donor groups which render it capable
of combining with metallic ions by chelation
[SOURCE: ISO 862:1984, 81]
3.7
specific activity
total radioactivity of the sample divided by its mass
Note 1 to entry: Specific activity is expressed in Bq/g.
[SOURCE: ISO 3925:2014, 3.4, modified — In the term, "activity" has been changed to "radioactivity"; Note 1
to entry has been added.]
4 Abbreviated terms
BFC bifunctional chelating agent
NP nanoparticle
TATE 1,4,8,11- tetraazacyclotetradecane-1,4,8,11-tetraacetic acid
CB-TE2A 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane
NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid
DOTA 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraaceticacid
DFO desferrioxamine
PET positron emission tomography
SPECT single photon emission computed tomography

5 Biodistribution study and radioisotopes
5.1 Biodistribution study
Biodistribution studies involve tracing the movement of materials of interest in an experimental animal or
[1][2]
human subject. For any medicinal product intended for human administration, whether in experimental
conditions during clinical trials or as an established treatment after approval by regulatory authorities,
extensive information is generated in anticipation of human administration to understand the potential
[3]
benefits and risks, as well as to anticipate and estimate the potential benefit-risk ratio profile. The
biodistribution of radiolabelled nanomaterials can be assessed using various methods, including image
quantification, tissue radioactivity measurement, or autoradiography.
Image-based biodistribution can be done by tracking the distribution of radiolabelled nanomaterials of
interest in an experimental animal or human subject using imaging techniques such as positron emission
tomography (PET), single-photon emission computed tomography (SPECT). This approach allows
researchers to visualize and quantify the spatial and temporal distribution of the nanomaterials of interest
[4]
in vivo, enabling them to better understand their pharmacokinetics and pharmacodynamics . Tissue-
based biodistribution is a preclinical research technique that involves the analysis of the distribution and
accumulation of radiolabelled nanomaterials in different tissues and organs of an animal or animal model.
This technique provides information on the pharmacokinetics and pharmacodynamics of nanomaterials,
which can include its absorption, distribution, metabolism and elimination (ADME) in different organs and
[5]
tissues. To perform a tissue-based biodistribution study, animals are typically treated with radiolabelled
nanomaterials. After a predetermined time, animals are sacrificed, and different tissues and organs are
collected and analysed for the presence and concentration of the drug. Whole-body slices can be made
to visualize the distribution of radioactivity in different organs and tissues using autoradiography. This
technique involves exposing the sliced tissue sections to a photographic film or imaging plate, which detects
the radioactive emissions and generates an image that can be analysed to determine the distribution of
the radiolabelled nanomaterial. Autoradiography is a widely used technique in preclinical research for
evaluating the biodistribution of radiolabelled nanomaterials, and it can provide valuable insights into how
[6]
the drug or nanomaterial is distributed throughout the body .
The administration route of nanomaterials, such as intravenous, inhalational, intratracheal, oral, or
intraperitoneal administration, can also be considered based on the intended clinical application. The
administration route is an important consideration when assessing the biodistribution of nanomaterials
since different routes can lead to distinct distribution patterns and pharmacokinetic profiles. Therefore,
researchers carefully select the appropriate method and administration route to obtain accurate and
reliable results.
5.2 Radioisotopes
Radioisotopes are isotopes that emit radiation and are commonly used for the diagnosis and treatment of
various human diseases. Diagnostic purposes make up about 90 % of radioisotope usage, while therapeutic
treatment makes up the remaining 10 %. Radioisotopes are labelled onto disease-targeting molecules,
which are then administered and targeted to specific organs or tissues through specific mechanisms. The
information from the radioisotopes is then collected and reconstructed by an imaging instrument to provide
information about disease localization and specific biological processes. Typically, diagnostic radioisotopes
are preferred for biodistribution studies because they have longer penetration depth and lower toxicity than
+ [7]
therapeutic radioisotopes. γ or β emitters are commonly used for this purpose.
The ability of γ rays to penetrate through the body depends on their energy, with higher energy leading
to higher penetration ratios. However, excessively high energy can decrease the detector's sensitivity and
resolution. As such, moderate-energy γ rays (between 30 keV and 300 keV) are optimal for γ camera or SPECT
[8][9] +
imaging. β particles can create two γ photons (each with an energy of 511 keV) via an annihilation
reaction, making them suitable for PET imaging. The range of a positron is influenced by its kinetic
[10]
energy, with lower kinetic energy leading to better imaging quality. When labelling nanomaterials with
radioisotopes, the physical half-life is also a critical factor to be considered because specific nanomaterials
have varying biological half-lives.

6 Radioisotope labelling methods for nanomaterials
6.1 General
The methods for radioisotope labelling and imaging of nanomaterials are crucial for various biomedical
applications. Two common approaches for radioisotope labelling are chelating agent-based (Figure 1, a) and
chelating agent-free (Figure 1, b to e) methods, as shown in the figure below.
Chelating agent-based methods involve the attachment of a chelating agent to the surface of the nanomaterial,
which then binds to a radioisotope. This approach provides stable binding and high labelling efficiency.
However, it can also result in the modification of the nanomaterial's properties and potential toxicity.
Chelating agent-free methods involve the direct binding of the radioisotope to the nanomaterial surface
without the use of a chelating agent. This approach preserves the nanomaterial's properties and reduces
potential toxicity. However, it can have lower labelling efficiency and stability.
Overall, the choice of labelling method depends on the specific application and desired properties of the
[11]
labelled nanomaterial.
a)  Chelator based b)  Radioactive + non-radioactive c)  Trapping
d)  Cation exchange
e)  Activated with high energy particle
Key
1 nanomaterial
2 radioactive nanomaterial
3 non-radioactive nanomaterial
4 high energy particle
5 radiometal
Figure 1 — Radioisotope labelling methods for nanomaterials

6.2 Pre- and post-surface labelling method
There are two approaches to radioisotope labelling of surface-modified nanomaterials, pre- and post-surface
[12]
labelling methods, as shown in Figure 2. The pre-surface-labelling method can be used for nanomaterials
that contain conjugation motifs on the surface, while the post-surface-labelling method can be used for
chelating agents on the surface. The radioisotope labelling procedure can involve harsh reaction conditions,
such as high/low pH, high temperature and/or the use of reducing or oxidizing agents. Therefore, the pre-
labelling method is a better choice for nanomaterials that are acid/base or high-temperature labile and
cannot meet such radioisotope labelling conditions directly.
Click chemistry, which was coined by the K. Barry Sharpless group in 2001, is a type of chemical reaction
[13]
that can be easily and rapidly achieved. It might be the best tool for the pre-surface labelling method.
Clickable nanomaterials can be labelled with radioisotope-labelled bifunctional chelating agents under
aqueous conditions and pH 7,4 at room temperature.
a) Pre-surface labelling
b) Post-surface labelling
Key
1 radioisotope
2 bifunctional chelating agent
Figure 2 — Schematic procedure of the pre- and post-surface-labelling methods
6.3 Chelating agent-based labelling
The chelating agent-based radioisotope labelling method involves the use of a bifunctional chelating agent
(BFC), such as 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TATE), 4,11-bis(carboxymethyl)-
1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A), (1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) or desferrioxamine (DFO), which coordinate
64 177 68 89 [14-17]
the metallic radioisotope including Cu, Lu, Ga and Zr, respectively (Figure 3). The BFC method
for radiolabelling has several advantages over other labelling methods. One advantage is that it provides
stable and high-affinity binding of the radioisotope to the nanomaterial surface. BFCs have a high affinity for
radioisotopes and can form stable complexes, which minimizes the risk of detachment of the radioisotope
from the nanomaterial surface. This method also enables high specific activity labelling, which means that
a high number of radioisotopes can be attached to a small amount of nanomaterial, resulting in improved
sensitivity and accuracy in biodistribution studies. Overall, the BFC method provides a reliable and versatile
[18]
approach for radiolabelling of nanomaterials for a variety of biomedical applications .

The BFC terminal carboxyl, aldehyde, amino, thiol, silanol, or isothiocyanate groups are commonly covalently
[19-21]
attached to the surface of nanoparticles (NPs) based on the coating layer applied during NP synthesis.
Although the chelating agent-based radioisotope labelling method has been widely used, the chemical
modification of NPs through BFC incorporation can have potential drawbacks. This is because the addition
of BFCs to NPs can alter their physical properties, such as size, surface charge and hydrophilicity, which can
[22]
subsequently affect the overall biodistribution and pharmacokinetic behaviour of the labelled NPs.
Figure 3 — Structures of most popular BFCs employed for complexation with metallic radioisotopes
In addition, selecting a suitable BFC to achieve optimal radioisotope labelling efficiency and stability poses
a significant challenge due to the potential release of metallic radioisotope from the BFC and transchelation
with endogenous proteins in vivo, resulting in suboptimal targeting that does not accurately represent
[23],[25]
the true biodistribution of radioisotope-labelled NPs. Additionally, the coordination chemistry of
different metallic radioisotopes varies significantly, so a chelating agent can work well with one metallic
72 69 45
radioisotope but not with others. Furthermore, certain metallic radioisotopes, such as As, Ge, and Ti,
still lack suitable chelating agents and radioisotope labelling methodologies. Therefore, new methods for
radiolabelling NPs are highly desirable for the advancement of radioisotope-labelled nanomaterials.

6.4 Chelating agent-free radioisotope labelling method
6.4.1 General
To better understand the intrinsic behaviour of NPs and to avoid the influence of BFC, chelating agent-free
labelling techniques have been developed. These methods eliminate the use of chelating agents and offer
a relatively simple, versatile and promising approach for radioisotope labelling of NPs using a variety of
[26]
metallic radioisotopes. The nonclassical chelating agent-free radioisotope labelling methods are mainly
[27]
divided into three categories and are discussed in detail by Goel et al. in a review.
6.4.2 Radioactive-plus-non-radioactive precursors
The nonclassical method for synthesizing radioactive NPs is also known as the radiochemical doping method.
This approach is relatively straightforward and involves adding a trace amount of a radioactive precursor
to the core of a non-radioactive precursor. The small amount of radioactive precursor incorporates into the
crystal lattice of the NPs, resulting in radioactive NPs that exhibit high stability and yield.
A wide variety of metallic radioisotopes can be used to synthesize radioisotope-labelled NPs. One example
64 [28]
of this type is [ Cu]CuS radioisotope-labelled NPs reported by Zhou et al., which were synthesized by
reacting radioactive CuCl , non-radioactive CuCl , and Na S in the presence of sodium citrate at 95 °C for
2 2 2
1 h. The average hydrodynamic particle size of the nanoparticle was 11 nm. The [ Cu]CuS NPs showed high
stability in various media in vitro with no agglomeration and no change in hydrodynamic particle size for up
to 7 d. Eventually, PEGylated [ Cu]CuS NPs showed high uptake in U87 human glioblastoma xenografts as a
result of enhanced permeability and retention effects, which subsequently increased the synergetic effect of
photothermal ablation therapy and radiotherapy at the target site.
[29]
Alvarez-Paneque et al. synthesized Cu nanoparticles using gold (Au) as a template with controlled size
and morphology. They prepared gold nanospheres with an average size of 57,0 ± 7,6 nm and gold nanorods
with a length of 65,7 ± 8,5 nm and thickness of 13,7 ± 2,3 nm, which were then coated with thiolated
polyethylene glycol (mPEG-SH). Cu was introduced to the Au crystal lattice after reduction with hydrazine
[30] 64
in the presence of poly(acrylic acid) as a stabilizer. Similarly, Sun et al. doped Cu on Au-NPs of different
sizes (10 nm, 30 nm and 80 nm) and shapes (sphere, rod and hexapod) using a similar methodology, which
was found to be radiochemically stable in vivo. PET images were performed after injecting [ Cu]Cu-Au
(80 nm) NPs and [ Cu]Cu-DOTA-Au (80 nm) NPs for comparison into mice via tail vein. At 1 h post-injection,
a negligible amount of activity was observed in the bladder of mice injected with [ Cu]Cu-Au (80 nm) NPs,
while a significant amount of activity was observed in the bladder of mice injected with [ Cu]Cu-DOTA-Au
(80 nm) NPs, suggesting that the chelating agent released Cu, which was excreted from the kidney. This
highlights the reliability and efficiency of the nonclassical and chelating agent-free method of radioisotope
labelling compared to chelating agent-based methods. This was further confirmed by injecting [ Cu]Cu-Au
NR808 and co-injecting RGD-[ Cu]Cu-Au NR808 with a blocking agent.
64 64
Another example of the hot and cold precursor method is the synthesis of [ Cu]CuAuNPs by allowing Cu
[31]
to directly enter the lattice of AuNPs, as reported by Zhou et al. In another study, glutathione-coated
near-infrared (NIR) emitting radioactive gold NPs (GS-[ Au]AuNPs) were synthesized by adding HAuCl
198 [32]
and H AuCl to a glutathione aqueous solution at 90 °C.
6.4.3 Specific trapping
Specific trapping involves the absorption, entrapment, or reaction of radioisotopes into specific sites of
NPs. This approach allows for fast and efficient direct radioisotope labelling of NPs without altering their
[33]
behaviour in vivo and yields highly stable radioisotope-labelled NPs. Lin et al. made an effort to load
RGD C, Cy5.5, and Cu simultaneously onto heavy-chain ferritins to produce nanoprobes for multimodal
imaging targeting integrin α β . Cy5.5 was chemically incorporated onto the ferritin surface, whereas
v 3
RGD D was introduced by amine coupling. Finally, bivalent Cu was loaded under acidic conditions by
exploiting the nanocage-like structure of ferritin. The ferritin cavity is capable of binding to bivalent Cu
with 60 % loading capacity, and free Cu was removed by a PD-10 column. It is believed that the hydrophilic
channels in ferritin, which connect the inside and outside of ferritins, serve as a path to allow ions to enter
the cavity. Once the ions enter the cavity of ferritin, they would find it hard to escape from it. It was found
that only 10 % of the activity was released over a period of 24 h after incubation in phosphate-buffered

saline and fetal bovine serum. In vivo PET images were acquired by injecting hybrid probes into U87MG
glioma tumour-bearing mice. The quantitative analysis showed continuous accumulation of activity in
the tumour up to 24 h post-injection (6,4 ± 1,7 % ID/g after 1 h, 7,5 ± 0,7 % ID/g after 4 h, 8,1 ± 0,1 % ID/g
after 24 h), which decreased to 7,5 ± 0,1 % ID/g at 40 h post-injection. The EPR (enhanced permeability and
retention) effect and active targeting were responsible for specific uptake in the tumour. The accumulation
in the tumour mediated by the receptor was confirmed by performing blocking experiments, which showed
that the uptake was significantly reduced.
[34] 64
Liu et al. reported on the successful radioisotope labelling of Cu to MoS -IO-(d)PEG with a moderate
radioisotope labelling yield of between 70 % to 85 % after 60 min of incubation at 37 °C. The Cu was
absorbed onto the surface of MoS and remained intact for up to 48 h in serum. The feasibility of using
[ Cu]Cu-MoS -IO-(d)PEG NPs for PET imaging was established in 4T1 tumour-bearing mice at various time
intervals. High accumulation and retention of [ Cu]Cu-MoS -IO-(d)PEG was observed in the tumour of 4T1
tumour-bearing mice at 3 h post-injection, which was retained until 24 h.
A number of reports have demonstrated that fluorine-18 ( F) can be labelled to rare-earth NPs with high
radiochemical yield and stability. Due to the short half-life of F (t = 110 min), the radioisotope labelling
1/2
procedures are necessarily simple, rapid, and efficient. In view of this, Li et al. synthesized tri-modal
18 18
F-labelled Na Bi O F :20 %Yb,0,5 %Tm NPs ([ F]UNBOF) through a facile inorganic reaction
0.20 0.80 0.35 1.91
between Na F, NH F, NaNO , Bi(NO ) , and Ln(NO ) (Ln = Yb/Tm) under vigorous stirring within 1 minute
4 3 3 3 3 3
18 18
at room temperature. Negligible dissociation of F was observed after incubating [ F]UNBOF NPs in fetal
18 [35]
bovine serum, indicating strong interaction of F with NPs.
Cui et al. synthesized trimodal Fe O @ NaYF core/shell inorganic nanoparticles for mapping lymph nodes
3 4 4
[36]
(LNs). Lanthanide elements Yb, Er, or Tm were doped into NaYF4 to achieve upconversion fluorescence,
while Co was doped into Fe O to optimize the magnetic properties of (Fe O @ NaYF (Yb, Tm) and
2 3 3 4 4
(Co . Fe O @NaYF (Yb, Er) NPs. The NPs' surfaces were modified by incorporating bisphosphonate
0 16 3 4 4
polyethylene glycol (BP-PEG) to improve their surface properties and solubility. Subsequently, F was
labelled onto the NPs with a radioisotope labelling yield of up to 38 % in just 5 min at room temperature.
The resulting 18F-labelled Co . Fe O @NaYF (Yb, Er)-BP-PEG and Fe O @ NaYF (Yb, Tm)-BP-PEG NPs were
0 16 3 4 4 3 4 4
found to be stable in human serum for up to 2 h, with 85 % of the activity remaining attached to the NPs.
The NPs' in vivo PET/MRI imaging capabilities were evaluated for the detection of popliteal LNs in response
to an acute inflammatory stimulus in the foot. A solution of F-labelled NPs was injected into the mouse's
footpad, and both popliteal and iliac LNs were visible in PET and MRI images 6 h post-injection.
6.4.4 Cation exchange
The cation exchange method is an alternative doping technique used for the synthesis of nanocrystals,
whereby the pre-existing cation in the nanomaterials is substituted with a different cation. This method is
promising as it allows for the production of NPs that cannot be synthesized directly, due to the fast reaction
kinetics at room temperature.
[37]
Recently, Tang et al. synthesized zinc sulfide (ZnS) quantum dot nanocrystals (QDs) using
3-mercaptopropionic acid as a capping agent. Thiol-functionalized polyethylene glycol (SH-PEG-OCH ) was
incorporated onto the surface of the QDs (QD-OCH ) to improve their stability and biocompatibility. The
68 64
QD-OCH was rapidly labelled with Ga or Cu within 15 min at 37 °C in a sodium acetate buffer with
68 64 64
radiochemical efficiency > 95 % for Ga and > 90 % for Cu. The Cu-labelled QD-OCH showed high
stability in a 1,8 mM DOTA and DTPA chelating agent solution in PBS, with most of the activity remaining
attached to QD-OCH . The stability against other metal ions, such as ferric chloride (1,8 mM), was also
tested, and the results showed that only a small portion (approximately 1,9 %) of radioactivity was released
68 64
from the Ga-labelled QD-OCH . In vivo PET imaging of [ Cu]Cu-QD-OCH in 4T1 mouse breast tumour
3 3
xenograft clearly showed tumour tissue at 3 h and 24 h post-injection scan. In contrast, the control group
[ Cu]Cu-DOTA showed only bladder uptake at 3 h post-injection, indicating rapid renal clearance.
[38] 64
Sun et al. developed a self-illuminating QD system by directly doping trace amounts of Cu into CdSe/
ZnS core/shell QDs via a cation-exchange reaction for luminescence and PET imaging. The Cu radioisotope
was labelled via cation exchange with 100 % radiochemical efficiency and showed high stability in fetal
bovine serum and mouse blood at 37 °C for 48 h. The Cu-labelled QDs were modified with amine-
polyethylene glycol-thiol (amine-PEG5000-thiol) to increase water solubility. The in vivo distribution of
Cu-doped QDs was studied in a U87MG glioblastoma xenograft model using PET. Mice were injected with

25 μg QD580 (250 µCi of Cu) via the tail vein, and PET scans were obtained at 1 h, 17 h, 24 h and 42 h post-
injection. Quantitative analysis (ROI) of the whole-body PET image showed 5 % ID/g uptake in the U87MG
glioblastoma tumour site, which further increased to 12 % ID/g at 17 h post-injection, and after 42 h, 10 %
ID/g of the QDs was still retained in the tumour.
6.4.5 Neutron or proton beam activation
This strategy involves the synthesis of nonradioactive NPs, which are then converted into radioactive NPs
through direct irradiation by a proton beam or thermal neutron.
[39] 152
Wang et al. synthesized carbon nanocapsules filled with nonradioactive Samarium-152 ( Sm) isotopes
152 152 153
( Sm@SWNT and Sm@MWNT) and then activated them through neutron irradiation to create Sm
for therapeutic use. The loading capacity of Sm was determined to be approximately 12,3 % mass
152 152
fraction for Sm@SWNT and 17,6 % mass fraction for Sm@MWNT, using inductively coupled plasma
14 −2 −1
mass spectrometry (ICP-MS). After irradiation in high neutron flux (1,6 × 10 n cm s ) and long neutron
153 153
irradiation time, high specific activities of [ Sm]Sm@ SWNT (6,33 GBq/mg) and [ Sm]Sm@ MWNT
153 153
(11,37 GBq/mg) were obtained. SPECT/CT imaging showed that both [ Sm]Sm@ SWNTs and [ Sm]Sm@
MWNT had similar biodistribution patterns, with activity mostly accumulating in spleen, lung and liver at
30 min post-injection and retaining up to 24 h post-injection. Quantitative γ-counting measurement validated
the distribution pattern of radioactivity. Additionally, the study demonstrated that the conjugates [ Sm]
Sm@ SWNTs and [ Sm]Sm@ MWNT were therapeutically effective in delaying the growth of metastatic
lung tumours.
Holmium-166 ( Ho) is a promising therapeutic radionuclide that emits high-energy β particles
(E = 1,84 MeV) and has a long half-life of 26,8 h. One way to prepare Ho-labelled nanoparticles (NPs)
max
is by neutron activation of the stable isotope Ho, which produces high specific activity. Di Pasqua et
[40] 165 165
al. have successfully prepared Ho-doped mesoporous silica type MCM-14 ( Ho-MSNs) NPs with a
particle size of 80 nm ‒ 100 nm in diameter. Subsequently, Ho-MSNs were irradiated in a reactor (1 MW)
12 2 166
at a thermal neutron flux of approximately 5,5 × 10 neutrons/cm s for 1 h to18 h to produce [ Ho]
Ho-MSNs. In a study, the biodistribution of [ Ho]Ho-MSNs was evaluated after intraperitoneal injection
in SKOV-3 ovarian tumour-bearing xenografts. The results showed high accumulation of the NPs in the
tumour (32,8 % ID/g ± 68,1 % ID/g) at 24 h post-injection, which increased to approximately 2,5 times
(81 % ID/g ± 7,5 % ID/g) at 1 week post-injection. This indicates that [ Ho]Ho-MSNs have the potential to
be used as an effective therapeutic agent for the treatment of ovarian tumours.
Proton beam activation of metal oxide NPs via (p,n), (p,α) nuclear reactions has been reported by Pérez
[41-43] [41] 18
Campaña et al. In one study, Pérez Campaña et al. prepared O-enriched aluminium oxide (Al O )
2 3
18 18
NPs in the presence of basic aqueous media, followed by activation via the O(p,n) F nuclear reaction with
a 16 MeV proton. The irradiation with 16 MeV did not significantly change the particle size of the Al O NPs,
2 3
confirming their high stability before and after irradiation.
[43] 18
In a follow up study Pérez-Campaña et al. prepared O-enriched titanium dioxide (TiO ) NPs by
18 18
bubbling NH (g) through an aqueous solution of TiCl in the presence of O-enriched water ([ O]H O) as
3 4 2
a solvent under an inert atmosphere to avoid incorporation of O and NH . The resulting TiO NPs were
3 2
irradiated with a 15 MeV proton for 6 min at a beam intensity of 5 µA. This converted the O-enriched
18 18 18 18
TiO NPs to F-labelled NPs via the O(p,α) F nuclear reaction, producing high amounts of F (≈ 700 kBq
−1
mg ) in a short irradiation time. This amount of radioactivity produced is sufficient for in vivo assessment
and distribution monitoring of NPs by PET/CT imaging. The F-labelled TiO NPs were administered
intravenously, and PET/CT images were obtained at different time intervals.
6.5 Dual radioisotope labelling
Dual radioisotope labelling is a technique that allows the fate of different components of a nanoparticle,
such as the inorganic core, the organic surface capping and the protein corona, to be traced individually.
Typically, this is achieved by labelling both the core and surface with different radioisotopes. This technique
enables the integrity of radioisotope labelling on the surface of nanomaterials to be evaluated.
[44] 197
Here is a typical example of dual isotope labelling using gold nanoparticle. The authors used Au, a
stable isotope, to create the gold nanoparticle, which was then stabilized by a ligand shell of dodecanethiol.
Following neutron activation, some of the Au atoms were converted into the radioactive isotope Au,

to form the radioisotope-labelled core. To make the nanoparticle water-soluble, a shell of the amphiphilic
polymer poly(isobutylene-alt-maleic anhydride)-graft-dodecyl was wrapped around the Au core. DOTA was
integrated into the polymer shell and loaded with indium (enriched with the radioisotope In), which acted
198 111
as the shell label. In this way, the nanoparticle core and shell were individually labelled by Au and In,
111 198
respectively. After administering [ In]In-DTPA-[ Au]gold nanoparticle into normal mice, the authors
traced the radioactivity using tissue-based biodistribution techniques. They found that the polymer shells of
polymer-coated Au nanoparticles were partially removed both in vitro and in vivo. In vivo, the nanoparticles
were mostly retained in the liver, and fragments of the organic shell were excreted through the kidneys. The
authors suggested that proteolytic enzymes present in these compartments caused a partial separation of
the organic shell from the inorganic core. They also noted that the amide bond linkage for connection of a
shell and BFC can cause the bond breakage by the enzyme. In contrast, using a different linkage, such as
[45]
thiourea, resulted in in vivo integrity after administration .
6.6 Choice of radioisotopes
The abundance of radioisotopes varies widely in terms of half-life and chemical properties, ranging from
alkali metals to lanthanides. Due to this diversity, it is important to select the appropriate radioisotope
for labelling nanomaterials for in vivo animal experiments. Characteristics such as decay half-life, decay
energy and availability of the isotope are also considered when choosing the ideal radioisotope for labelling
68 99m 188
(see Annex A). Commonly used radioisotopes include generator-produced Ga, Tc or Re; cyclotron-
18 64 89 111 123 124 125 177
produced F, Cu, Zr, In, I or I; and reactor-produced I or Lu. For in vivo biodistribution
studies, radioisotopes that emit γ-rays are typically used. Positron-emitting radioisotopes also emit γ-rays
after the annihilation reaction. The physical half-life of radioisotopes is also a significant consideration.
18 68 99m
Radioisotopes with short half-lives, such as F (t = 109,8 min), Ga (t = 67,7 min) and Tc (t = 6 h),
1/2 1/2 1/2
64 111 89
or those with longer half-lives, such as Cu (t = 12,7 h), In (t = 67 h), and Zr (t = 78,4 h), are
1/2 1/2 1/2
chosen based on the biodistribution study's purpose. The physical half-life of a radioisotope can be matched
with the biological half-life of the nanomaterials being labelled with these radioisotopes to allow them to
reach the tar
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