# [18F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques

Maëlle Deleuziere, Éric Benoist, Isabelle Quelven, Emmanuel Gras, Catherine Amiens

► To cite this version:

Maëlle Deleuziere, Éric Benoist, Isabelle Quelven, Emmanuel Gras, Catherine Amiens. [18F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques. Molecules, 2024, 29 (7), pp.1537. 10.3390/molecules29071537 hal-04536242

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# [18F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques

Maëlle Deleuziere, Éric Benoist, Isabelle Quelven, Emmanuel Gras, Catherine Amiens

► To cite this version:

Maëlle Deleuziere, Éric Benoist, Isabelle Quelven, Emmanuel Gras, Catherine Amiens. [18F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques. Molecules, 2024, 29 (7), pp.1537. 10.3390/molecules29071537 hal-04536242

HAL Id: hal-04536242

https://hal.science/hal-04536242v1

Submitted on 8 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Review

# [¹⁸F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques

Maëlle Deleuziere ¹,²,³ D, Éric Benoist ¹ D, Isabelle Quelven ² D, Emmanuel Gras ⁴ D and Catherine Amiens ³,* D

1 SPCMIB, CNRS UMR 5068, Université de Toulouse III Paul Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France; maelle.deleuziere@univ-tlse3.fr (M.D.); eric.benoist@univ-tlse3.fr (É.B.)

2 Toulouse NeuroImaging Center (ToNIC), INSERM/UPS UMR 1214, University Hospital of Toulouse-Purpan, CEDEX 3, 31024 Toulouse, France; quelven-bertin.i@chu-toulouse.fr

3 LCC-CNRS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France

4 Laboratoire Hétérochimie Fondamentale et Appliquée, UMR 5069, CNRS—Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France; emmanuel.gras@univ-tlse3.fr

* Correspondence: catherine.amiens@lcc-toulouse.fr

**Abstract:** A wide range of nano-objects is found in many applications of our everyday life. Recognition of their peculiar properties and ease of functionalization has prompted their engineering into multifunctional platforms that are supposed to afford efficient tools for the development of biomedical applications. However, bridging the gap between bench to bedside cannot be expected without a good knowledge of their behaviour in vivo, which can be obtained through non-invasive imaging techniques, such as positron emission tomography (PET). Their radiolabelling with [¹⁸F]-fluorine, a technique already well established and widely used routinely for PET imaging, with [¹⁸F]-FDG for example, and in preclinical investigation using [¹⁸F]-radiolabelled biological macromolecules, has, therefore, been developed. In this context, this review highlights the various nano-objects studied so far, the reasons behind their radiolabelling, and main in vitro and/or in vivo results obtained thereof. Then, the methods developed to introduce the radioelement are presented. Detailed indications on the chemical steps involved are provided, and the stability of the radiolabelling is discussed. Emphasis is then made on the techniques used to purify and analyse the radiolabelled nano-objects, a point that is rarely discussed despite its technical relevance and importance for accurate imaging. The pros and cons of the different methods developed are finally discussed from which future work can develop.

**Keywords:** nanoparticle; fluorine-18; radiofluorination; conjugation; characterization; purification; PET imaging; multimodal imaging

## 1. Introduction

Nanoparticles can be found in a large range of morphologies (sizes, shapes, porosity, etc.), and present a large panel of physico-chemical properties (lipophilicity, surface charges, optical and magnetic properties, etc.). Their large surface over volume ratio enables the grafting of a variety of functional groups at their surface, which can further modulate the properties of the nanoparticles or endorse them with new complementary properties. They are, therefore, seen as versatile nanoplatforms that can be tailored at will depending on the application envisaged. Especially in nanomedicine, the possibility to adjust their surface state to modulate their uptake by the reticuloendothelial system and to incorporate targeting agents has prompted their investigation as imaging probes, therapeutic tools, and even for theranostic applications, among others. Approaches based on nanoparticles can also be used to modulate the biodistribution and bioavailability of molecular therapeutic agents in order to improve their therapeutic index.

<figure><img src="image_2.png" /></figure>

check for
updates

**Citation:** Deleuziere, M.; Benoist, É.; Quelven, I.; Gras, E.; Amiens, C. [¹⁸F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques.

Molecules **2024**, *29*, 1537.
https://doi.org/10.3390/molecules29071537

Academic Editor: Carlos Geraldes

Received: 28 February 2024

Revised: 20 March 2024

Accepted: 26 March 2024

Published: 29 March 2024

<figure><img src="image_3.png" /></figure>

**Copyright:** © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Review

# [¹⁸F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques

Maëlle Deleuziere ¹,²,³ D, Éric Benoist ¹ D, Isabelle Quelven ² D, Emmanuel Gras ⁴ D and Catherine Amiens ³,* D

¹ SPCMIB, CNRS UMR 5068, Université de Toulouse III Paul Sabatier, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France; maelle.deleuziere@univ-tlse3.fr (M.D.); eric.benoist@univ-tlse3.fr (É.B.)

² Toulouse NeuroImaging Center (ToNIC), INSERM/UPS UMR 1214, University Hospital of Toulouse-Purpan, CEDEX 3, 31024 Toulouse, France; quelven-bertin.i@chu-toulouse.fr

³ LCC-CNRS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France

⁴ Laboratoire Hétérochimie Fondamentale et Appliquée, UMR 5069, CNRS—Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France; emmanuel.gras@univ-tlse3.fr

* Correspondence: catherine.amiens@lcc-toulouse.fr

**Abstract:** A wide range of nano-objects is found in many applications of our everyday life. Recognition of their peculiar properties and ease of functionalization has prompted their engineering into multifunctional platforms that are supposed to afford efficient tools for the development of biomedical applications. However, bridging the gap between bench to bedside cannot be expected without a good knowledge of their behaviour in vivo, which can be obtained through non-invasive imaging techniques, such as positron emission tomography (PET). Their radiolabelling with [¹⁸F]-fluorine, a technique already well established and widely used routinely for PET imaging, with [¹⁸F]-FDG for example, and in preclinical investigation using [¹⁸F]-radiolabelled biological macromolecules, has, therefore, been developed. In this context, this review highlights the various nano-objects studied so far, the reasons behind their radiolabelling, and main in vitro and/or in vivo results obtained thereof. Then, the methods developed to introduce the radioelement are presented. Detailed indications on the chemical steps involved are provided, and the stability of the radiolabelling is discussed. Emphasis is then made on the techniques used to purify and analyse the radiolabelled nano-objects, a point that is rarely discussed despite its technical relevance and importance for accurate imaging. The pros and cons of the different methods developed are finally discussed from which future work can develop.

**Keywords:** nanoparticle; fluorine-18; radiofluorination; conjugation; characterization; purification; PET imaging; multimodal imaging

## 1. Introduction

Nanoparticles can be found in a large range of morphologies (sizes, shapes, porosity, etc.), and present a large panel of physico-chemical properties (lipophilicity, surface charges, optical and magnetic properties, etc.). Their large surface over volume ratio enables the grafting of a variety of functional groups at their surface, which can further modulate the properties of the nanoparticles or endorse them with new complementary properties. They are, therefore, seen as versatile nanoplatforms that can be tailored at will depending on the application envisaged. Especially in nanomedicine, the possibility to adjust their surface state to modulate their uptake by the reticuloendothelial system and to incorporate targeting agents has prompted their investigation as imaging probes, therapeutic tools, and even for theranostic applications, among others. Approaches based on nanoparticles can also be used to modulate the biodistribution and bioavailability of molecular therapeutic agents in order to improve their therapeutic index.

<figure><img src="image_2.png" /></figure>

check for
updates

**Citation:** Deleuziere, M.; Benoist, É.; Quelven, I.; Gras, E.; Amiens, C. [¹⁸F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques.

Molecules **2024**, *29*, 1537.
https://doi.org/10.3390/molecules29071537

Academic Editor: Carlos Geraldes

Received: 28 February 2024

Revised: 20 March 2024

Accepted: 26 March 2024

Published: 29 March 2024

<figure><img src="image_3.png" /></figure>

**Copyright:** © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
However, despite a large number of in vitro and preclinical studies reporting on nanoparticles, the number of nanotherapeutics that reach clinical use remains relatively low. Several barriers exist that limit the effective preclinical evaluation and clinical translation of nanoparticles to therapy. Key barriers include an insufficient understanding of the nanoparticles' in vivo biodistribution. This is associated with an inadequate translation from animal models to humans, as well as a high inter- and intra-patient variability. This is particularly illustrated in the Enhanced Permeability and Retention (EPR) effect in tumours [1,2]. Translational and clinical studies depend on the ability to access relevant information on nanoparticle pharmacokinetics, tissue accumulation, and therapy outcome in vivo. This critical information can be obtained owing to non-invasive imaging techniques, which are powerful tools to visualize nanoparticles in vivo, and hence bridge the gap between benchtop and bedside.

A large number of imaging techniques are available including magnetic resonance imaging (MRI), X-ray computed tomography (CT), and optical molecular imaging techniques, such as near-infrared fluorescence (NIRF) imaging or fluorescence molecular tomography (FMT), as well as nuclear imaging by positron emission tomography (PET) and single-photon emission computed tomography (SPECT). CT and MRI are both very powerful anatomical imaging techniques but exhibit poor sensitivity for visualizing exogenously administered contrast agents, requiring the administration of large amounts of exogenous agents. MRI has been employed to track nanoparticles in vivo by functionalizing them with paramagnetic (manganese, gadolinium) or superparamagnetic (iron oxide) contrast agents. However, given its poor sensitivity, rather large doses should be injected. Moreover, quantification is not straightforward, as the generated contrast not only depends on the agent's concentration but is also affected by other factors, such as relaxivity, tissue properties, and magnetic field inhomogeneities. Optical molecular imaging techniques can provide valuable information on nanoparticles' in vivo biodistribution. Nevertheless, emitted light's limited tissue penetration, fluorophore bleaching and quenching, or tissue absorption hamper a proper quantification of the signal. Conversely, PET and SPECT are very sensitive, as they allow the detection of sub-pM tracer concentrations and enable accurate signal quantification [3]. Given the low amounts of imaging agent needed, the biological processes under study are, therefore, assumed to be undisturbed. Quantitative information about pharmacokinetic parameters, biodistribution profiles, or target site accumulation of nanoparticles can be obtained. So, during clinical evaluation, nuclear imaging can be used to select personalized treatment for patients. PET and SPECT, which provide functional images, are combined with anatomical imaging techniques, such as CT and lately also MRI, in hybrid imaging scanners to improve resolution and obtain truly quantitative data corrected for tissue photon attenuation.

While providing reliable information, SPECT is progressively replaced by the more accurately quantitative PET. Exact attenuation correction, precise scatter correction, and high sensitivity (combined with the possibility to perform true whole-body imaging in a reasonable time) constitute the key factors for the superiority of PET over SPECT.

Employing nuclear imaging to investigate nanoparticles in vivo behaviour requires their radiolabelling. A range of PET emitters are now routinely available with short-lived radionuclides, such as gallium-68 (67.7 min), fluorine-18 (110 min) or copper-64 (12.7 h), or long half-life ones, particularly iodine-124 (4.18 d) and zirconium-89 (78.4 h).

In addition to the radionuclide half-life, a large number of parameters must be taken into account to select a radionuclide. Among the factors of choice, cost, scalability, radiolabelling strategies, radiochemical yield and purity, and the reaction's duration are key factors to consider.

In most targeted radiometal-based radiopharmaceuticals, the metal is bound to a pharmacophore by a bifunctional chelating agent (BFCA), which forms a stable covalent linkage between the label and the targeting ligand (vector) and ensures the stable complexation of the metal in vivo [4]. In many instances, the chelator and the vector are connected via a spacer moiety to separate the individual components of the conjugate to avoid potential
However, despite a large number of in vitro and preclinical studies reporting on nanoparticles, the number of nanotherapeutics that reach clinical use remains relatively low. Several barriers exist that limit the effective preclinical evaluation and clinical translation of nanoparticles to therapy. Key barriers include an insufficient understanding of the nanoparticles' in vivo biodistribution. This is associated with an inadequate translation from animal models to humans, as well as a high inter- and intra-patient variability. This is particularly illustrated in the Enhanced Permeability and Retention (EPR) effect in tumours [1,2]. Translational and clinical studies depend on the ability to access relevant information on nanoparticle pharmacokinetics, tissue accumulation, and therapy outcome in vivo. This critical information can be obtained owing to non-invasive imaging techniques, which are powerful tools to visualize nanoparticles in vivo, and hence bridge the gap between benchtop and bedside.

A large number of imaging techniques are available including magnetic resonance imaging (MRI), X-ray computed tomography (CT), and optical molecular imaging techniques, such as near-infrared fluorescence (NIRF) imaging or fluorescence molecular tomography (FMT), as well as nuclear imaging by positron emission tomography (PET) and single-photon emission computed tomography (SPECT). CT and MRI are both very powerful anatomical imaging techniques but exhibit poor sensitivity for visualizing exogenously administered contrast agents, requiring the administration of large amounts of exogenous agents. MRI has been employed to track nanoparticles in vivo by functionalizing them with paramagnetic (manganese, gadolinium) or superparamagnetic (iron oxide) contrast agents. However, given its poor sensitivity, rather large doses should be injected. Moreover, quantification is not straightforward, as the generated contrast not only depends on the agent's concentration but is also affected by other factors, such as relaxivity, tissue properties, and magnetic field inhomogeneities. Optical molecular imaging techniques can provide valuable information on nanoparticles' in vivo biodistribution. Nevertheless, emitted light's limited tissue penetration, fluorophore bleaching and quenching, or tissue absorption hamper a proper quantification of the signal. Conversely, PET and SPECT are very sensitive, as they allow the detection of sub-pM tracer concentrations and enable accurate signal quantification [3]. Given the low amounts of imaging agent needed, the biological processes under study are, therefore, assumed to be undisturbed. Quantitative information about pharmacokinetic parameters, biodistribution profiles, or target site accumulation of nanoparticles can be obtained. So, during clinical evaluation, nuclear imaging can be used to select personalized treatment for patients. PET and SPECT, which provide functional images, are combined with anatomical imaging techniques, such as CT and lately also MRI, in hybrid imaging scanners to improve resolution and obtain truly quantitative data corrected for tissue photon attenuation.

While providing reliable information, SPECT is progressively replaced by the more accurately quantitative PET. Exact attenuation correction, precise scatter correction, and high sensitivity (combined with the possibility to perform true whole-body imaging in a reasonable time) constitute the key factors for the superiority of PET over SPECT.

Employing nuclear imaging to investigate nanoparticles in vivo behaviour requires their radiolabelling. A range of PET emitters are now routinely available with short-lived radionuclides, such as gallium-68 (67.7 min), fluorine-18 (110 min) or copper-64 (12.7 h), or long half-life ones, particularly iodine-124 (4.18 d) and zirconium-89 (78.4 h).

In addition to the radionuclide half-life, a large number of parameters must be taken into account to select a radionuclide. Among the factors of choice, cost, scalability, radiolabelling strategies, radiochemical yield and purity, and the reaction's duration are key factors to consider.

In most targeted radiometal-based radiopharmaceuticals, the metal is bound to a pharmacophore by a bifunctional chelating agent (BFCA), which forms a stable covalent linkage between the label and the targeting ligand (vector) and ensures the stable complexation of the metal in vivo [4]. In many instances, the chelator and the vector are connected via a spacer moiety to separate the individual components of the conjugate to avoid potential
interference. Chelators contain several functional groups for coordination to the radiometal of choice. In contrast, non-metal radionuclides are linked to the vector by a covalent bond, directly or via a linker [5]. The versatility and applicability of covalently bound radionuclides are enormous, especially as the chemical structure is not altered [¹¹C], or only slightly [¹⁸F], compared with PET tracers based on a radiometal.

Fluorine exists in nature only as one stable isotope, namely [¹⁹F], with a 100% abundance. [¹⁸F], the radioactive isotope of fluorine, is easily accessible since it is produced in cyclotron from inexpensive precursors. It exhibits some peculiar physical properties such as (i) a highly pure decay by emission of a positron of relatively low energy (meaning high positron yield, small mean free path, and thus good resolution and signal over noise ratio); (ii) a half-life time of 110 min, inducing an acceptable dosimetry but also leaving enough time to perform chemistry and imaging. This imaging relies on the annihilation event between the emitted positron and one electron from the surrounding matter, releasing two γ photons of 511 keV at 180°, which are detected by the camera within a short timeframe. This allows to precisely determine the location of the annihilation event and thus the position of the [¹⁸F] decay. This radionuclide is easily accessible since it is produced in a cyclotron from inexpensive precursors and a large number of methods are available to bind [¹⁸F] to vectors. All these properties have promoted an extensive use of [¹⁸F] for years and make it one of the most frequently applied radionuclides in diagnosis [6].

Hence, based on a careful selection of original works found in the commonly accessed databases (SciFinder, Web of Science, PubMed, etc.), this work builds a critical review of the [¹⁸F]-radiolabelling of nanoplatforms. Section 2 outlines the nanoplatforms studied so far. They are either inert, to be used as carriers (such as polymers, liposomes, porous silica, etc.), or/and intrinsically active (iron-based oxides, gold, gadolinium-based oxides nanoparticles, etc.) to associate complementary diagnostics or therapeutic tools in the same object. From a chemical point of view, two main categories arise: organic nanoplatforms (block-copolymers, dendrimers, liposomes, etc.), and inorganic ones (which can be classified according to their core and surface chemical features). Section 3 provides the reader with detailed information on the various prosthetic groups used for radiofluorination, which are mentioned in the first chapter. The methods and techniques used to purify and analyse the radiolabelled nanoplatforms are then reviewed in Section 4 (Figure 1). Based on this overview, we then discuss the scope and limitations of the use of [¹⁸F]-labelled nanoplatforms and the expectations of their future development toward translational applications. At the end of the document, a list of abbreviations and definitions is provided to ease the reading for researchers from different fields.

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Overview of this report's structure created with Figma.</figcaption></figure>
interference. Chelators contain several functional groups for coordination to the radiometal of choice. In contrast, non-metal radionuclides are linked to the vector by a covalent bond, directly or via a linker [5]. The versatility and applicability of covalently bound radionuclides are enormous, especially as the chemical structure is not altered [¹¹C], or only slightly [¹⁸F], compared with PET tracers based on a radiometal.

Fluorine exists in nature only as one stable isotope, namely [¹⁹F], with a 100% abundance. [¹⁸F], the radioactive isotope of fluorine, is easily accessible since it is produced in cyclotron from inexpensive precursors. It exhibits some peculiar physical properties such as (i) a highly pure decay by emission of a positron of relatively low energy (meaning high positron yield, small mean free path, and thus good resolution and signal over noise ratio); (ii) a half-life time of 110 min, inducing an acceptable dosimetry but also leaving enough time to perform chemistry and imaging. This imaging relies on the annihilation event between the emitted positron and one electron from the surrounding matter, releasing two γ photons of 511 keV at 180°, which are detected by the camera within a short timeframe. This allows to precisely determine the location of the annihilation event and thus the position of the [¹⁸F] decay. This radionuclide is easily accessible since it is produced in a cyclotron from inexpensive precursors and a large number of methods are available to bind [¹⁸F] to vectors. All these properties have promoted an extensive use of [¹⁸F] for years and make it one of the most frequently applied radionuclides in diagnosis [6].

Hence, based on a careful selection of original works found in the commonly accessed databases (SciFinder, Web of Science, PubMed, etc.), this work builds a critical review of the [¹⁸F]-radiolabelling of nanoplatforms. Section 2 outlines the nanoplatforms studied so far. They are either inert, to be used as carriers (such as polymers, liposomes, porous silica, etc.), or/and intrinsically active (iron-based oxides, gold, gadolinium-based oxides nanoparticles, etc.) to associate complementary diagnostics or therapeutic tools in the same object. From a chemical point of view, two main categories arise: organic nanoplatforms (block-copolymers, dendrimers, liposomes, etc.), and inorganic ones (which can be classified according to their core and surface chemical features). Section 3 provides the reader with detailed information on the various prosthetic groups used for radiofluorination, which are mentioned in the first chapter. The methods and techniques used to purify and analyse the radiolabelled nanoplatforms are then reviewed in Section 4 (Figure 1). Based on this overview, we then discuss the scope and limitations of the use of [¹⁸F]-labelled nanoplatforms and the expectations of their future development toward translational applications. At the end of the document, a list of abbreviations and definitions is provided to ease the reading for researchers from different fields.

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Overview of this report's structure created with Figma.</figcaption></figure>
## 2. [$^{18}$F]-Radiolabelled Nanoplatforms

### 2.1. *Organic Nanoplatforms*

The first organic nanoplatforms investigated were natural macromolecules, such as albumin. They display a well-defined molecular structure, and their radiolabelling is now well mastered. This section will focus on statistical organic nano-objects, the physico-chemical descriptors of which, such as composition, size (geometrical and hydrodynamical) or surface charge, are only known as average values with standard deviations. Furthermore, some of these nano-objects (micelles, droplets, etc.) are highly dynamic nanostructures in which the physico-chemical properties depend on pH and ionic strength, among other biological parameters.

#### 2.1.1. Fluxional Micelles, Nanoemulsions, and Bubbles

*Perfluoro gas bubbles* encapsulated in self-assembled lipids, and albumin, were developed for ultrasound imaging. A higher ligand density at the surface of the bubbles was determined when albumin was used for their stabilization [7]. After functionalization with anti-VEGFR2 antibody, labelling of the albumin shell with [$^{18}$F]-succinimidylfluorobenzoate ([$^{18}$F]-SFB 38, see Section 3.2.3) allowed to study their trafficking in vivo by both PET and echography. Given their size (from 0.5 to 6 µm, with an average hydrodynamic diameter of 1.98 µm), these bubbles were easily trapped in the lungs, then accumulated in the liver. Lower accumulation in liver was achieved upon conjugation with anti-VEGFR2 antibody to target breast cancer (17.7% ID/g compared to 41.8% ID/g). This illustrated the potential of such bubbles for the delivery of therapeutic agents to cancer tumours (ultrasound-assisted therapy).

*Perfluorocarbon (PFCD) droplets*, also used in echography, further exhibit exquisite properties for $^{19}$F MRI and as drug delivery vehicles [8,9]. Nanoemulsions of perfluorohexane stabilized by a perfluoro-surfactant (average size 175 nm) and nanoemulsions of perfluorooctylbromide stabilized by lipids (average size 260 nm) were radiolabelled to investigate their biodistribution in normal mice by PET [10]. This evidenced different tissue uptake and clearance pathways for the two nanoemulsions.

*Nanoemulsions* of vitamin E stabilized by sphingomyelin and stearylamine are a recent class of drug delivery vehicles suited for the transport of lipophilic drugs (e.g., curcumin and resveratrol) [11]. Their ease of modification allows incorporation of vectors, and functionalization towards [$^{18}$F]-labelling. For example, the lipophilic C₁₈ chain of SH-PEG₁₂-C₁₈ and RPMpeptide-PEG₈-C₁₈ (RPM peptide = CPIEDRPMC) allowed their integration into the nanoemulsion. Given their amphiphilic nature, they serve as additional surfactants affording surface RPM peptide as a vector and SH functions for further derivatization. The labelling process with [$^{18}$F]-FBEM (4-[$^{18}$F]fluorobenzamido-N-ethylmaleimide, see Section 3.3.2) was successfully accomplished utilizing the SH groups exposed at the surface of the nanoemulsion droplets. This nano-object (hydrodynamic size = 149 nm, ζ potential = +37 mV) produces a detectable signal for PET imaging 2 h post-injection. The pharmacokinetics and excretion routes were analysed through ex vivo biodistribution and in vivo imaging studies both in healthy and metastatic colorectal tumour-grafted mice. The results confirmed the benefits of surface decoration with RPM to target metastatic tumours, which were already observed with RPM-modified chitosan-stearic micelles [12,13].

*Self-assembly of amphiphilic molecules* into direct micelles allows the encapsulation of lipophilic drugs. However, micelles form only when the concentration of the amphiphile is above a certain threshold (the critical micellar concentration, c.m.c) and their stability depends on ionic strength and pH. Their persistence in vivo and thus their usefulness as delivery vehicles has been questioned, promoting the development of their radiolabelling to investigate their fate in vivo.

As an example, polyethylene glycol (PEG) 3400-distearoylphosphatidylethanolamine (DSPE) spontaneously form micelles (at concentrations above 10 µM) of approx. 10 nm diameter [14]. These micelles were tested as potential cargo for [$^{18}$F]-dasatinib in comparison to phosphatidyl:cholesterol liposomes (see Section 2.1.1). After loading, the average
## 2. [$^{18}$F]-Radiolabelled Nanoplatforms

### 2.1. *Organic Nanoplatforms*

The first organic nanoplatforms investigated were natural macromolecules, such as albumin. They display a well-defined molecular structure, and their radiolabelling is now well mastered. This section will focus on statistical organic nano-objects, the physico-chemical descriptors of which, such as composition, size (geometrical and hydrodynamical) or surface charge, are only known as average values with standard deviations. Furthermore, some of these nano-objects (micelles, droplets, etc.) are highly dynamic nanostructures in which the physico-chemical properties depend on pH and ionic strength, among other biological parameters.

#### 2.1.1. Fluxional Micelles, Nanoemulsions, and Bubbles

*Perfluoro gas bubbles* encapsulated in self-assembled lipids, and albumin, were developed for ultrasound imaging. A higher ligand density at the surface of the bubbles was determined when albumin was used for their stabilization [7]. After functionalization with anti-VEGFR2 antibody, labelling of the albumin shell with [$^{18}$F]-succinimidylfluorobenzoate ([$^{18}$F]-SFB 38, see Section 3.2.3) allowed to study their trafficking in vivo by both PET and echography. Given their size (from 0.5 to 6 µm, with an average hydrodynamic diameter of 1.98 µm), these bubbles were easily trapped in the lungs, then accumulated in the liver. Lower accumulation in liver was achieved upon conjugation with anti-VEGFR2 antibody to target breast cancer (17.7% ID/g compared to 41.8% ID/g). This illustrated the potential of such bubbles for the delivery of therapeutic agents to cancer tumours (ultrasound-assisted therapy).

*Perfluorocarbon (PFCD) droplets*, also used in echography, further exhibit exquisite properties for $^{19}$F MRI and as drug delivery vehicles [8,9]. Nanoemulsions of perfluorohexane stabilized by a perfluoro-surfactant (average size 175 nm) and nanoemulsions of perfluorooctylbromide stabilized by lipids (average size 260 nm) were radiolabelled to investigate their biodistribution in normal mice by PET [10]. This evidenced different tissue uptake and clearance pathways for the two nanoemulsions.

*Nanoemulsions* of vitamin E stabilized by sphingomyelin and stearylamine are a recent class of drug delivery vehicles suited for the transport of lipophilic drugs (e.g., curcumin and resveratrol) [11]. Their ease of modification allows incorporation of vectors, and functionalization towards [$^{18}$F]-labelling. For example, the lipophilic C₁₈ chain of SH-PEG₁₂-C₁₈ and RPMpeptide-PEG₈-C₁₈ (RPM peptide = CPIEDRPMC) allowed their integration into the nanoemulsion. Given their amphiphilic nature, they serve as additional surfactants affording surface RPM peptide as a vector and SH functions for further derivatization. The labelling process with [$^{18}$F]-FBEM (4-[$^{18}$F]fluorobenzamido-N-ethylmaleimide, see Section 3.3.2) was successfully accomplished utilizing the SH groups exposed at the surface of the nanoemulsion droplets. This nano-object (hydrodynamic size = 149 nm, ζ potential = +37 mV) produces a detectable signal for PET imaging 2 h post-injection. The pharmacokinetics and excretion routes were analysed through ex vivo biodistribution and in vivo imaging studies both in healthy and metastatic colorectal tumour-grafted mice. The results confirmed the benefits of surface decoration with RPM to target metastatic tumours, which were already observed with RPM-modified chitosan-stearic micelles [12,13].

*Self-assembly of amphiphilic molecules* into direct micelles allows the encapsulation of lipophilic drugs. However, micelles form only when the concentration of the amphiphile is above a certain threshold (the critical micellar concentration, c.m.c) and their stability depends on ionic strength and pH. Their persistence in vivo and thus their usefulness as delivery vehicles has been questioned, promoting the development of their radiolabelling to investigate their fate in vivo.

As an example, polyethylene glycol (PEG) 3400-distearoylphosphatidylethanolamine (DSPE) spontaneously form micelles (at concentrations above 10 µM) of approx. 10 nm diameter [14]. These micelles were tested as potential cargo for [$^{18}$F]-dasatinib in comparison to phosphatidyl:cholesterol liposomes (see Section 2.1.1). After loading, the average
micellar size increased to 50 nm. Loading densities of [¹⁸F]-dasatinib [drug/lipid (mol/mol) ratio] were found to be 1/233 and 1/2000 (mol/mol) for micelles and liposomes, respectively. PET investigations showed that the uptake in tumours (glioma-bearing mice), was significantly higher for drug-loaded micelles compared to liposomes. This result was explained by the smaller size of the micelles in comparison to that of the liposomes (120 nm). Local concentrations of the amphiphile were estimated to be close to that of the c.m.c., suggesting the easy release of the drug in vivo.

Triglyceride-rich lipoprotein self-assemblies (chylomicron nanoparticles) that display a lipophilic core have inspired the development of chylomicron-like radiotracers. For example, encapsulation of a fluorescent, [¹⁸F]-radiolabelled fatty acid inside chylomicron nanoparticles (average geometrical size 156 nm, and hydrodynamic diameter 164 nm) was performed. It allowed to study the brown adipose tissue in insulin-resistant conditions by a combination of fluorescence and PET imaging and generally outperformed [¹⁸F]-FDG [15].

Inorganic nanoparticles have also been encapsulated inside micelles to improve their biocompatibility and pharmacokinetics. Thus, encapsulation of radiolabelled THCPSi nanoparticles has been studied [16]. In an improved approach, late radiolabelling of the surfactant of the micelles encapsulating quantum dots was also investigated [17].

Self-assembly of radiolabelled macromolecules enhances their in vivo imaging by PET owing to high [¹⁸F] concentration. This is exemplified by the investigation of the biodistribution of [¹⁸F]-hyperbranched polyether ligands **1** (Figure 2). Their self-assembly into micelles led to locally high concentration of [¹⁸F]. PET imaging showed the rapid renal excretion of these polyethers with full clearance reached after 1 h [18]. This feature is a high added value when efficient self-assembly uptake by tumour cells is achieved, [19,20] and it was recently applied to detect and monitor apoptosis in vivo [21].

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Topological description of the hyperbranched polyether.</figcaption></figure>

*Liposomes* are vesicles formed by the self-assembly of phospholipids, such as **2** and **3**, into spherical bilayers (see Figure 3) or onion-like multilayers. A wide range of phospholipids, natural or synthetic, can be used to afford liposomes of different properties (size, rigidity, fluidity, permeability, stability, and electrical charge). A surfactant can be added to the liposome formulation to increase its deformability. Cholesterol **4** is generally added to increase their fluidity (Figure 3c). As cell membrane mimics, liposomes are considerably more robust nano-objects than micelles. They can accommodate a large range of hydrophilic (in their aqueous core) or hydrophobic (in the lipidic bilayer) molecules.
micellar size increased to 50 nm. Loading densities of [¹⁸F]-dasatinib [drug/lipid (mol/mol) ratio] were found to be 1/233 and 1/2000 (mol/mol) for micelles and liposomes, respectively. PET investigations showed that the uptake in tumours (glioma-bearing mice), was significantly higher for drug-loaded micelles compared to liposomes. This result was explained by the smaller size of the micelles in comparison to that of the liposomes (120 nm). Local concentrations of the amphiphile were estimated to be close to that of the c.m.c., suggesting the easy release of the drug in vivo.

Triglyceride-rich lipoprotein self-assemblies (chylomicron nanoparticles) that display a lipophilic core have inspired the development of chylomicron-like radiotracers. For example, encapsulation of a fluorescent, [¹⁸F]-radiolabelled fatty acid inside chylomicron nanoparticles (average geometrical size 156 nm, and hydrodynamic diameter 164 nm) was performed. It allowed to study the brown adipose tissue in insulin-resistant conditions by a combination of fluorescence and PET imaging and generally outperformed [¹⁸F]-FDG [15].

Inorganic nanoparticles have also been encapsulated inside micelles to improve their biocompatibility and pharmacokinetics. Thus, encapsulation of radiolabelled THCPSi nanoparticles has been studied [16]. In an improved approach, late radiolabelling of the surfactant of the micelles encapsulating quantum dots was also investigated [17].

Self-assembly of radiolabelled macromolecules enhances their in vivo imaging by PET owing to high [¹⁸F] concentration. This is exemplified by the investigation of the biodistribution of [¹⁸F]-hyperbranched polyether ligands **1** (Figure 2). Their self-assembly into micelles led to locally high concentration of [¹⁸F]. PET imaging showed the rapid renal excretion of these polyethers with full clearance reached after 1 h [18]. This feature is a high added value when efficient self-assembly uptake by tumour cells is achieved, [19,20] and it was recently applied to detect and monitor apoptosis in vivo [21].

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Topological description of the hyperbranched polyether.</figcaption></figure>

*Liposomes* are vesicles formed by the self-assembly of phospholipids, such as **2** and **3**, into spherical bilayers (see Figure 3) or onion-like multilayers. A wide range of phospholipids, natural or synthetic, can be used to afford liposomes of different properties (size, rigidity, fluidity, permeability, stability, and electrical charge). A surfactant can be added to the liposome formulation to increase its deformability. Cholesterol **4** is generally added to increase their fluidity (Figure 3c). As cell membrane mimics, liposomes are considerably more robust nano-objects than micelles. They can accommodate a large range of hydrophilic (in their aqueous core) or hydrophobic (in the lipidic bilayer) molecules.
Liposomes can also be modified by incorporation of various polymers with lipidic segments in the membrane (Figure 3d). For example, linear or hyperbranched PEG polymers are frequently added to improve their pharmacokinetic profiles [22,23]. Their targeting can also be envisaged by the same method. Given their ease of preparation and functionalization, they are the first nano-carriers used for clinical applications and their development largely contributed to the development of nanomedicine. They are now found in many clinical applications [24,25]. These developments have necessitated the study of their trafficking in vivo. Many studies made use of radiolabelling for this purpose [26,27]. Table 1 reports some typical use of their [18F]-labelling. Three modes of introduction of the radiolabelled probes can be envisaged for the liposomes: (1) introducing a radiofluorinated cargo in the core; (2) introducing a radiolabelled unit in the bilayer (either a labelled cholesterol derivative or another radiolabelled hydrophobic derivative); and (3) labelling functional groups at the surface of the liposome. The location of the radiolabel is also given in Table 1.

<figure><img src="image_6.png" /><figcaption>**Figure 3.** (a) Structural illustration of glycerolphospholipid. R₁ and R₂ can be saturated or unsaturated fatty acids, such as decanoic acid, lauric acid, palmitic acid, oleic acid, myristic acid, stearic acid, and erucic acid. R₃ can be phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, phosphatidylglycerol, and cardiolipin; (b) structure of sphingomyelin; (c) structure of cholesterol; and (d) schematic representation of a bilayer liposome and possible surface modifications (created with Biorender).</figcaption></figure>
Liposomes can also be modified by incorporation of various polymers with lipidic segments in the membrane (Figure 3d). For example, linear or hyperbranched PEG polymers are frequently added to improve their pharmacokinetic profiles [22,23]. Their targeting can also be envisaged by the same method. Given their ease of preparation and functionalization, they are the first nano-carriers used for clinical applications and their development largely contributed to the development of nanomedicine. They are now found in many clinical applications [24,25]. These developments have necessitated the study of their trafficking in vivo. Many studies made use of radiolabelling for this purpose [26,27]. Table 1 reports some typical use of their [18F]-labelling. Three modes of introduction of the radiolabelled probes can be envisaged for the liposomes: (1) introducing a radiofluorinated cargo in the core; (2) introducing a radiolabelled unit in the bilayer (either a labelled cholesterol derivative or another radiolabelled hydrophobic derivative); and (3) labelling functional groups at the surface of the liposome. The location of the radiolabel is also given in Table 1.

<figure><img src="image_6.png" /><figcaption>**Figure 3.** (a) Structural illustration of glycerolphospholipid. R₁ and R₂ can be saturated or unsaturated fatty acids, such as decanoic acid, lauric acid, palmitic acid, oleic acid, myristic acid, stearic acid, and erucic acid. R₃ can be phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, phosphatidylglycerol, and cardiolipin; (b) structure of sphingomyelin; (c) structure of cholesterol; and (d) schematic representation of a bilayer liposome and possible surface modifications (created with Biorender).</figcaption></figure>
<table><caption>**Table 1.** Selected examples of [¹⁸F]-labelled liposomes and purposes of the studies (NR: data not reported).</caption><thead><tr><th>Constituents</th><th>Approximate<br>Hydrodynamic<br>Diameters (nm)</th><th>ζ Potential<br>(mV)</th><th>Radiolabelling<br>Location (Bilayer,<br>Core, or Surface)</th><th>Objective of the Study</th><th>Ref.</th></tr></thead><tbody><tr><td>PC: cholesterol</td><td>120</td><td>NR</td><td>Core</td><td>Evaluation of<br>[¹⁸F]-dasatinib uptake in<br>glioblastoma as a function<br>of formulation to optimize<br>dosage</td><td>[14]</td></tr><tr><td>DOPC: cholesterol plus<br>surface coating (PEG or<br>hyperbranched PG)</td><td>408, 92, and 168<br>for liposomes<br>non-modified,<br>modified with<br>PEG, and<br>modified with<br>hyperbranched<br>PG, respectively</td><td>NR</td><td>Bilayer with<br>cholestene<br>surface with<br>PEGylated and<br>hyperbranched<br>polyethers</td><td>Comparison of the effect<br>of hyperbranched PG<br>surface coating to the one<br>of linear PEG on the<br>biodistribution and<br>pharmacokinetics of<br>liposomes</td><td>[18,23]</td></tr><tr><td>DOPC: cholesterol<br>modified with<br>hyperbranched PEG<sub>38</sub> and<br>trimannose</td><td>109</td><td>NR</td><td>Surface</td><td>PET assessment of<br>trimannose as a liposome<br>vector to target dendritic<br>cells</td><td>[28]</td></tr><tr><td>DSPC: cholesterol</td><td>90, 170, and 570<br>depending on<br>formulation</td><td>NR</td><td>Bilayer</td><td>Study of labelling method<br>efficiency and liposomal<br>trafficking</td><td>[29]</td></tr><tr><td>DPPC: cholesterol:<br>DSPE-PEG<sub>2000</sub></td><td>45</td><td>NR</td><td>Bilayer</td><td>Study of liposome<br>trafficking</td><td>[30]</td></tr><tr><td>Hydrogenated soybean<br>phosphatidylcholine:<br>cholesterol: stearic acid,<br>surface modified by<br>PEG<sub>5000</sub>-DSPE</td><td>200–250</td><td>NR</td><td>Bilayer</td><td>Study of the delivery of<br>oxygen into ischematic<br>brain by<br>liposome-encapsulated<br>hemoglobin</td><td>[31]</td></tr><tr><td>DOTPA: cholesterol</td><td>218</td><td>+36</td><td>Core</td><td>Study of the effect of<br>liposome encapsulation<br>on the biodistribution of<br>siRNA</td><td>[32]</td></tr><tr><td>DSPC: cholesterol, surface<br>modified by DSPE-<br>PEG<sub>2000</sub></td><td>100</td><td>-2.9 ± 1.4</td><td>Bilayer</td><td>Study of liposomal drug<br>delivery systems and if<br>they could enter the<br>ischemic brain region and<br>be a therapeutic strategy<br>for ischemic stroke<br>treatment</td><td>[33]</td></tr><tr><td>DSPC: cholesterol, surface<br>modified by DSPE-<br>PEG<sub>2000</sub>, DSPE-PEG-TCO,<br>and pHLIP</td><td>143</td><td>NR</td><td>Bilayer</td><td>Study of targeting by<br>pHLIP and if it improved<br>accumulation into<br>tumours</td><td>[34]</td></tr><tr><td>DPPC: DSPE-PEG<sub>2000</sub><br>targeted by arginine-rich<br>peptides</td><td>From 70 to 200,<br>depending on<br>peptide loading<br>and formulation</td><td>From -48 to<br>+42, depending<br>on peptide<br>loading and<br>formulation</td><td>Bilayer</td><td>Study of the effect of the<br>presence and structure of<br>targeting peptides on the<br>adhesion of the<br>formulation on blood<br>vessel walls in the heart</td><td>[35]</td></tr></tbody></table>
<table><caption>**Table 1.** Selected examples of [¹⁸F]-labelled liposomes and purposes of the studies (NR: data not reported).</caption><thead><tr><th>Constituents</th><th>Approximate<br>Hydrodynamic<br>Diameters (nm)</th><th>ζ Potential<br>(mV)</th><th>Radiolabelling<br>Location (Bilayer,<br>Core, or Surface)</th><th>Objective of the Study</th><th>Ref.</th></tr></thead><tbody><tr><td>PC: cholesterol</td><td>120</td><td>NR</td><td>Core</td><td>Evaluation of<br>[¹⁸F]-dasatinib uptake in<br>glioblastoma as a function<br>of formulation to optimize<br>dosage</td><td>[14]</td></tr><tr><td>DOPC: cholesterol plus<br>surface coating (PEG or<br>hyperbranched PG)</td><td>408, 92, and 168<br>for liposomes<br>non-modified,<br>modified with<br>PEG, and<br>modified with<br>hyperbranched<br>PG, respectively</td><td>NR</td><td>Bilayer with<br>cholestene<br>surface with<br>PEGylated and<br>hyperbranched<br>polyethers</td><td>Comparison of the effect<br>of hyperbranched PG<br>surface coating to the one<br>of linear PEG on the<br>biodistribution and<br>pharmacokinetics of<br>liposomes</td><td>[18,23]</td></tr><tr><td>DOPC: cholesterol<br>modified with<br>hyperbranched PEG<sub>38</sub> and<br>trimannose</td><td>109</td><td>NR</td><td>Surface</td><td>PET assessment of<br>trimannose as a liposome<br>vector to target dendritic<br>cells</td><td>[28]</td></tr><tr><td>DSPC: cholesterol</td><td>90, 170, and 570<br>depending on<br>formulation</td><td>NR</td><td>Bilayer</td><td>Study of labelling method<br>efficiency and liposomal<br>trafficking</td><td>[29]</td></tr><tr><td>DPPC: cholesterol:<br>DSPE-PEG<sub>2000</sub></td><td>45</td><td>NR</td><td>Bilayer</td><td>Study of liposome<br>trafficking</td><td>[30]</td></tr><tr><td>Hydrogenated soybean<br>phosphatidylcholine:<br>cholesterol: stearic acid,<br>surface modified by<br>PEG<sub>5000</sub>-DSPE</td><td>200–250</td><td>NR</td><td>Bilayer</td><td>Study of the delivery of<br>oxygen into ischematic<br>brain by<br>liposome-encapsulated<br>hemoglobin</td><td>[31]</td></tr><tr><td>DOTPA: cholesterol</td><td>218</td><td>+36</td><td>Core</td><td>Study of the effect of<br>liposome encapsulation<br>on the biodistribution of<br>siRNA</td><td>[32]</td></tr><tr><td>DSPC: cholesterol, surface<br>modified by DSPE-<br>PEG<sub>2000</sub></td><td>100</td><td>-2.9 ± 1.4</td><td>Bilayer</td><td>Study of liposomal drug<br>delivery systems and if<br>they could enter the<br>ischemic brain region and<br>be a therapeutic strategy<br>for ischemic stroke<br>treatment</td><td>[33]</td></tr><tr><td>DSPC: cholesterol, surface<br>modified by DSPE-<br>PEG<sub>2000</sub>, DSPE-PEG-TCO,<br>and pHLIP</td><td>143</td><td>NR</td><td>Bilayer</td><td>Study of targeting by<br>pHLIP and if it improved<br>accumulation into<br>tumours</td><td>[34]</td></tr><tr><td>DPPC: DSPE-PEG<sub>2000</sub><br>targeted by arginine-rich<br>peptides</td><td>From 70 to 200,<br>depending on<br>peptide loading<br>and formulation</td><td>From -48 to<br>+42, depending<br>on peptide<br>loading and<br>formulation</td><td>Bilayer</td><td>Study of the effect of the<br>presence and structure of<br>targeting peptides on the<br>adhesion of the<br>formulation on blood<br>vessel walls in the heart</td><td>[35]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Constituents</th><th>Approximate<br>Hydrodynamic<br>Diameters (nm)</th><th>ζ Potential<br>(mV)</th><th>Radiolabelling<br>Location (Bilayer,<br>Core, or Surface)</th><th>Objective of the Study</th><th>Ref.</th></tr></thead><tbody><tr><td>DSPC: cholesterol, surface<br>modified by PEG and<br>targeted by a peptide<br>(APRPG)</td><td>100</td><td>NR</td><td>Bilayer</td><td>Radiotracer delivery to<br>image brain cancer<br>tumours</td><td>[36]</td></tr><tr><td>DSPC: cholesterol surface<br>modified by<br>DSPE-PEG<sub>2000</sub></td><td>NR</td><td>NR</td><td>Bilayer</td><td>Validation of an<br>automated preparation<br>method of radiolabelled<br>liposomes, and study of<br>liposome trafficking<br>(tumour accumulation)</td><td>[37]</td></tr><tr><td>Cholestero l:<br>sphingomyelin:<br>mal-PEG-PE<br>functionalized by<br>phosphatidic acid or a<br>lipidic derivate of<br>TEG-curcumin</td><td>133 or 237 for<br>liposomes with<br>phosphatidic<br>acid or lipidic<br>derivate of<br>TEG-curcumin,<br>respectively</td><td>NR</td><td>Bilayer with<br>radiofluorinated<br>lipids<br>Core with<br>radiolabelled<br>curcumin analogs</td><td>Evaluation of ability of<br>functionalized liposomes<br>to cross the blood–brain<br>barrier and bind to the<br>amyloid β plaque</td><td>[38]</td></tr><tr><td>DPPC: cholesterol:<br>DSPE-PEG2000 surface<br>modified by<br>DPPE-DTPA-[<sup>111</sup>In]</td><td>168</td><td>-2.3</td><td>Core</td><td>Development of a dual<br>[<sup>18</sup>F] (PET)/<sup>111</sup>In (SPECT)<br>radio imaging tool</td><td>[39]</td></tr></tbody></table>

DOPC = 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE-DTPA = 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N diethylenetriaminepentaacetic acid; DOTPA = 1,2-dioleoyl-3-trimethylammonium-propane; DSPC = distearoylphosphatidylcholine; mal-PEG-PE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt; PEG = polyethyleneglycol; DSPE-PEG = N-[monomethoxy-polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine; PG = polyglycerol; pHLIP = pH (Low) Insertion Peptide; SiRNA = small interfering RNA; TCO = trans-cyclooctene; and TEG = triethyleneglycol.

### 2.1.2. Polymers

Another approach to develop nano-carriers is to form rigid micelles by a careful design of biocompatible block-copolymers integrating both hydrophilic and lipophilic segments in their structure. [<sup>18</sup>F]-labelling has been implemented to study their biodistribution in vivo and especially to check the efficiency of their targeting prior to their application as drug delivery cargo. Block-copolymers studied so far comprise four main categories of polymeric segments: poly-N-(2-hydroxypropyl) methacrylamide (pHPMA), polyesters, polyethers, and polyoxazolines (POX).

#### Poly-N-(2-Hydroxypropyl)Methacrylamide (pHPMA)-Based Macromolecules

pHPMA (Figure 4) is a non-immunogenic, nontoxic, and usually long-circulating polymer, which spontaneously forms micelles in physiological conditions and allows effective drug delivery. It is already applied in clinical practice [22]. Its conjugation to folic acid was investigated as a possible tool to target cancer cells. Study of the biodistribution of [<sup>18</sup>F]-pHPMA and [<sup>18</sup>F]-pHPMA-folate 5 showed a distinct difference between passive and active targeting, with a faster uptake for the targeted polymer independent of its size (10.5 kDa or 52.5 kDa) [40]. Copolymerization to yield random HMPA/LMA 6 (LMA = laurylmethacrylate) copolymers, or block-copolymers pHPMA-pLMA, was studied to impart lipophilicity to the macromolecule and facilitate its use for the delivery of lipophilic drugs [41,42]. All these copolymers formed stable micelles leading to nanomaterials with hydrodynamic radii of 30–40 nm (random copolymers) to 59–112 nm (block-copolymers) with high stability in serum. After [<sup>18</sup>F]-labelling, the biodistribution of copolymers with different molecular weights (from ca 13 to 134 kDa), structures (random
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th>Constituents</th><th>Approximate<br>Hydrodynamic<br>Diameters (nm)</th><th>ζ Potential<br>(mV)</th><th>Radiolabelling<br>Location (Bilayer,<br>Core, or Surface)</th><th>Objective of the Study</th><th>Ref.</th></tr></thead><tbody><tr><td>DSPC: cholesterol, surface<br>modified by PEG and<br>targeted by a peptide<br>(APRPG)</td><td>100</td><td>NR</td><td>Bilayer</td><td>Radiotracer delivery to<br>image brain cancer<br>tumours</td><td>[36]</td></tr><tr><td>DSPC: cholesterol surface<br>modified by<br>DSPE-PEG<sub>2000</sub></td><td>NR</td><td>NR</td><td>Bilayer</td><td>Validation of an<br>automated preparation<br>method of radiolabelled<br>liposomes, and study of<br>liposome trafficking<br>(tumour accumulation)</td><td>[37]</td></tr><tr><td>Cholestero l:<br>sphingomyelin:<br>mal-PEG-PE<br>functionalized by<br>phosphatidic acid or a<br>lipidic derivate of<br>TEG-curcumin</td><td>133 or 237 for<br>liposomes with<br>phosphatidic<br>acid or lipidic<br>derivate of<br>TEG-curcumin,<br>respectively</td><td>NR</td><td>Bilayer with<br>radiofluorinated<br>lipids<br>Core with<br>radiolabelled<br>curcumin analogs</td><td>Evaluation of ability of<br>functionalized liposomes<br>to cross the blood–brain<br>barrier and bind to the<br>amyloid β plaque</td><td>[38]</td></tr><tr><td>DPPC: cholesterol:<br>DSPE-PEG2000 surface<br>modified by<br>DPPE-DTPA-[<sup>111</sup>In]</td><td>168</td><td>-2.3</td><td>Core</td><td>Development of a dual<br>[<sup>18</sup>F] (PET)/<sup>111</sup>In (SPECT)<br>radio imaging tool</td><td>[39]</td></tr></tbody></table>

DOPC = 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE-DTPA = 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N diethylenetriaminepentaacetic acid; DOTPA = 1,2-dioleoyl-3-trimethylammonium-propane; DSPC = distearoylphosphatidylcholine; mal-PEG-PE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt; PEG = polyethyleneglycol; DSPE-PEG = N-[monomethoxy-polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine; PG = polyglycerol; pHLIP = pH (Low) Insertion Peptide; SiRNA = small interfering RNA; TCO = trans-cyclooctene; and TEG = triethyleneglycol.

### 2.1.2. Polymers

Another approach to develop nano-carriers is to form rigid micelles by a careful design of biocompatible block-copolymers integrating both hydrophilic and lipophilic segments in their structure. [<sup>18</sup>F]-labelling has been implemented to study their biodistribution in vivo and especially to check the efficiency of their targeting prior to their application as drug delivery cargo. Block-copolymers studied so far comprise four main categories of polymeric segments: poly-N-(2-hydroxypropyl) methacrylamide (pHPMA), polyesters, polyethers, and polyoxazolines (POX).

#### Poly-N-(2-Hydroxypropyl)Methacrylamide (pHPMA)-Based Macromolecules

pHPMA (Figure 4) is a non-immunogenic, nontoxic, and usually long-circulating polymer, which spontaneously forms micelles in physiological conditions and allows effective drug delivery. It is already applied in clinical practice [22]. Its conjugation to folic acid was investigated as a possible tool to target cancer cells. Study of the biodistribution of [<sup>18</sup>F]-pHPMA and [<sup>18</sup>F]-pHPMA-folate 5 showed a distinct difference between passive and active targeting, with a faster uptake for the targeted polymer independent of its size (10.5 kDa or 52.5 kDa) [40]. Copolymerization to yield random HMPA/LMA 6 (LMA = laurylmethacrylate) copolymers, or block-copolymers pHPMA-pLMA, was studied to impart lipophilicity to the macromolecule and facilitate its use for the delivery of lipophilic drugs [41,42]. All these copolymers formed stable micelles leading to nanomaterials with hydrodynamic radii of 30–40 nm (random copolymers) to 59–112 nm (block-copolymers) with high stability in serum. After [<sup>18</sup>F]-labelling, the biodistribution of copolymers with different molecular weights (from ca 13 to 134 kDa), structures (random
or block), and monomer ratios was studied first in healthy rats. The difference was better observed in high molecular weight polymers, with the block-copolymers being much more retained in the liver and spleen than the random ones. Passive targeting of tumours was further assessed, with results being model dependant. The macromolecule architecture had no impact on its accumulation in AT1 prostate carcinoma, but random copolymers were shown to accumulate more than the block-copolymers in Walker-256 mammary carcinoma.

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Poly-N-(2-hydroxypropyl)methacrylamide-based macromolecules.</figcaption></figure>

More recently, pHPMA was used as a [¹⁸F]-nitroimidazole tracer delivery cargo. Thus, 7 exhibits a linear pHPMA chain, which was [¹⁸F]-labelled with a pH sensitive linker. The study showed the specific release of the [¹⁸F]-radiotracer in the vicinity of cancer tumours that present lower pH. So, by this means, the uptake of the radiotracer in cancer cells, which grow in acidic conditions, was facilitated [43].

## Polyester-Based Macromolecules

The polyester segments are often associated to polyether or polyamide segments (Figure 5) to form amphiphilic structures that self-assemble into micelles with low c.m.c. value. The slow biodegradation of the polyester block ensures a continuous delivery of the drug at the site of interest when a proper targeting vector is installed at the surface of the polymeric micelles.

Poly(lactic-co-glycolic acid) (PLGA 8, see Figure 5) nanoparticles are recognized as drug delivery vehicles capable of transporting a wide range of therapeutic agents (small molecules, proteins, and oligonucleotides, etc.) while protecting them from degradation and clearance. With proper surface modification of the PLGA nanoparticles, their diffusion in the extracellular matrix of the brain has been tuned (in rats) [44]. Given the fast distribution of the nanoparticles in vivo (from minutes to a few hours), radiolabelling with the short-live [¹⁸F] radioisotope was developed to minimise radiation exposure. Two sizes of polymeric nanoparticles were investigated (147 (±27) nm and 71 (±13) nm geometrical diameters). Nanoparticles were delivered directly and continuously to the rat striatum and their diffusion was monitored by PET. It confirmed that the diffusion region was always spherical, with a larger volume reached by the smaller particles.
or block), and monomer ratios was studied first in healthy rats. The difference was better observed in high molecular weight polymers, with the block-copolymers being much more retained in the liver and spleen than the random ones. Passive targeting of tumours was further assessed, with results being model dependant. The macromolecule architecture had no impact on its accumulation in AT1 prostate carcinoma, but random copolymers were shown to accumulate more than the block-copolymers in Walker-256 mammary carcinoma.

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Poly-N-(2-hydroxypropyl)methacrylamide-based macromolecules.</figcaption></figure>

More recently, pHPMA was used as a [¹⁸F]-nitroimidazole tracer delivery cargo. Thus, 7 exhibits a linear pHPMA chain, which was [¹⁸F]-labelled with a pH sensitive linker. The study showed the specific release of the [¹⁸F]-radiotracer in the vicinity of cancer tumours that present lower pH. So, by this means, the uptake of the radiotracer in cancer cells, which grow in acidic conditions, was facilitated [43].

## Polyester-Based Macromolecules

The polyester segments are often associated to polyether or polyamide segments (Figure 5) to form amphiphilic structures that self-assemble into micelles with low c.m.c. value. The slow biodegradation of the polyester block ensures a continuous delivery of the drug at the site of interest when a proper targeting vector is installed at the surface of the polymeric micelles.

Poly(lactic-co-glycolic acid) (PLGA 8, see Figure 5) nanoparticles are recognized as drug delivery vehicles capable of transporting a wide range of therapeutic agents (small molecules, proteins, and oligonucleotides, etc.) while protecting them from degradation and clearance. With proper surface modification of the PLGA nanoparticles, their diffusion in the extracellular matrix of the brain has been tuned (in rats) [44]. Given the fast distribution of the nanoparticles in vivo (from minutes to a few hours), radiolabelling with the short-live [¹⁸F] radioisotope was developed to minimise radiation exposure. Two sizes of polymeric nanoparticles were investigated (147 (±27) nm and 71 (±13) nm geometrical diameters). Nanoparticles were delivered directly and continuously to the rat striatum and their diffusion was monitored by PET. It confirmed that the diffusion region was always spherical, with a larger volume reached by the smaller particles.
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Polyester-based macromolecules.</figcaption></figure>

A block-copolymer 9 comprising a polyester block (1,8-octanediol/glutaric acid) and a polyether PEG1500 block was designed and studied as a delivery vehicle for the hydrophobic anti-cancer drug Paclitaxel. The particles obtained from this polymeric structure displayed sizes below 200 nm with very low polydispersity index and negative $ζ$ potential (−30 mV). The PEG block is expected to be exposed at the surface of the nanoparticles while the more hydrophobic polyester block accommodates the Paclitaxel in the core of the nanoparticle. After i.v. injection to rats, a low uptake in liver and renal clearance of the nanoparticles was observed by PET for nanoparticles exposing PEG chains at their surface, as expected [45].

Micelles of polylactic acid/polysarcosine (PS-PLA) block-copolymer 10 have also been evaluated as a potential radiotracer for in vivo PET imaging of solid tumours [46]. In this self-assembly, the sarcosine block of the polymers constitutes the hydrophilic, external part of the micelle. Once labelled, a hydrodynamic size of 41.5 nm was measured. Accumulation of the radiotracer in the tumour of HeLa-bearing mice was monitored by PET. A high tumour/muscle signal ratio was evidenced, which was associated to the EPR effect, as well as a low tumour/blood ratio that was associated to the high stability of the micelles in the blood stream and to their slow clearance from blood.

A comparative study was carried out on a series of block-copolymers of various structures: D-polylactide segments grafted on chitosan (PDLA-CS 11, positively charged in physiological conditions), PEG modified poly(lactic-co-glycolic acid)-b-polylysine (PEG-PLGA-POLLS 12, also positively charged), and PEG-modified phosphatidylserine/calcium phosphate hybrids (PEG-PS-CaP 13, negatively charged). (Figure 5) These block-copolymers self-assembled into micelles with hydrodynamic sizes and $ζ$ potential values of 120 nm and +16.5 mV, 130 nm and +12.5 mV, and 100 nm and −12.5 mV, respectively [47]. Their biodistribution was followed by PET after injection in tumour-bearing mice. PET imaging revealed the higher impact of the PEG modification over surface charges, which allowed a faster accumulation of the nanoparticles in the tumour and longer residence time in the blood stream.

### Poly(2-Alkyl-2-Oxazoline)s (POX)

POX 14 (Figure 6) are recognized as promising alternatives to PEG with good bio-compatibility and stealth. Their easy synthesis by ring-opening polymerization allows the formation of block co-polymers from different oxazoline monomers, and hence a fine
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Polyester-based macromolecules.</figcaption></figure>

A block-copolymer **9** comprising a polyester block (1,8-octanediol/glutaric acid) and a polyether PEG1500 block was designed and studied as a delivery vehicle for the hydrophobic anti-cancer drug Paclitaxel. The particles obtained from this polymeric structure displayed sizes below 200 nm with very low polydispersity index and negative $ζ$ potential (−30 mV). The PEG block is expected to be exposed at the surface of the nanoparticles while the more hydrophobic polyester block accommodates the Paclitaxel in the core of the nanoparticle. After i.v. injection to rats, a low uptake in liver and renal clearance of the nanoparticles was observed by PET for nanoparticles exposing PEG chains at their surface, as expected [45].

Micelles of polylactic acid/polysarcosine (PS-PLA) block-copolymer **10** have also been evaluated as a potential radiotracer for in vivo PET imaging of solid tumours [46]. In this self-assembly, the sarcosine block of the polymers constitutes the hydrophilic, external part of the micelle. Once labelled, a hydrodynamic size of 41.5 nm was measured. Accumulation of the radiotracer in the tumour of HeLa-bearing mice was monitored by PET. A high tumour/muscle signal ratio was evidenced, which was associated to the EPR effect, as well as a low tumour/blood ratio that was associated to the high stability of the micelles in the blood stream and to their slow clearance from blood.

A comparative study was carried out on a series of block-copolymers of various structures: D-polylactide segments grafted on chitosan (PDLA-CS **11**, positively charged in physiological conditions), PEG modified poly(lactic-co-glycolic acid)-b-polylysine (PEG-PLGA-POLL **12**, also positively charged), and PEG-modified phosphatidylserine/calcium phosphate hybrids (PEG-PS-CaP **13**, negatively charged). (Figure 5) These block-copolymers self-assembled into micelles with hydrodynamic sizes and $ζ$ potential values of 120 nm and +16.5 mV, 130 nm and +12.5 mV, and 100 nm and −12.5 mV, respectively [47]. Their biodistribution was followed by PET after injection in tumour-bearing mice. PET imaging revealed the higher impact of the PEG modification over surface charges, which allowed a faster accumulation of the nanoparticles in the tumour and longer residence time in the blood stream.

### Poly(2-Alkyl-2-Oxazoline)s (POX)

POX **14** (Figure 6) are recognized as promising alternatives to PEG with good bio-compatibility and stealth. Their easy synthesis by ring-opening polymerization allows the formation of block co-polymers from different oxazoline monomers, and hence a fine
tuning of the properties of the final macromolecule. Especially, its hydrophobicity can be adjusted by varying the length of the alkyl residue on the 2-oxazoline monomer.

<figure><img src="image_9.png" /><figcaption>**Figure 6.** Polyoxazoline-based macromolecules.</figcaption></figure>

For example, heptyl- and pentynyl-substituted oxazoline monomers were used to prepare a block-copolymer where the alkyne pendant groups were used to cross-link the polymer chains and stabilize the nanoparticles formed [48]. Furthermore, an organosilicon fluoride acceptor could be used as the initiator in the ROP, allowing in fine the radiolabelling of the nanoparticles by isotope exchange (using a silicon-fluoride acceptor (SiFA) [¹⁸F]-labelling). Nanoparticles with distinct hydrodynamic diameters (20, 33, 45, and 72 nm) were prepared [49]. Their passive uptake in a murine mammary tumour model were studied depending on particle size. It showed that the 33 nm large nanoparticles outperformed the others, exhibiting the best tumour to muscle ratio.

## Miscellaneous

Ring-opening olefin metathesis was applied to a set of two norbornene imides (one with a hydrophobic cinnamoyl group for photocross-linking, and one with a hydrophilic PEG$_{600}$ substituent for biocompatibility) to produce amphiphilic co-polymers **15** with precisely controlled molecular weight (Figure 7). Cross-linking was carried out to stabilize the micelles [50]. Micelles of four different geometrical sizes in the range 13 to 40 nm, and hydrodynamic sizes in the range 47 to 142 nm, respectively, were prepared. Their labelling by [¹⁸F] was efficiently achieved, offering perspectives for PET imaging, albeit no in vitro nor in vivo study was reported.

<figure><img src="image_10.png" /><figcaption>**Figure 7.** Polynorbornene imide-based macromolecules.</figcaption></figure>

### 2.1.3. Dendrimers and Hyperbranched Molecules

Dendrimers are hyperbranched polymers that grow concentrically from a multifunctional core. Each starting branch corresponds to a dendron, itself ramified into new branches (Figure 8).
tuning of the properties of the final macromolecule. Especially, its hydrophobicity can be adjusted by varying the length of the alkyl residue on the 2-oxazoline monomer.

<figure><img src="image_9.png" /><figcaption>**Figure 6.** Polyoxazoline-based macromolecules.</figcaption></figure>

For example, heptyl- and pentynyl-substituted oxazoline monomers were used to prepare a block-copolymer where the alkyne pendant groups were used to cross-link the polymer chains and stabilize the nanoparticles formed [48]. Furthermore, an organosilicon fluoride acceptor could be used as the initiator in the ROP, allowing in fine the radiolabelling of the nanoparticles by isotope exchange (using a silicon–fluoride acceptor (SiFA) [¹⁸F]-labelling). Nanoparticles with distinct hydrodynamic diameters (20, 33, 45, and 72 nm) were prepared [49]. Their passive uptake in a murine mammary tumour model were studied depending on particle size. It showed that the 33 nm large nanoparticles outperformed the others, exhibiting the best tumour to muscle ratio.

## Miscellaneous

Ring-opening olefin metathesis was applied to a set of two norbornene imides (one with a hydrophobic cinnamoyl group for photocross-linking, and one with a hydrophilic PEG$_{600}$ substituent for biocompatibility) to produce amphiphilic co-polymers **15** with precisely controlled molecular weight (Figure 7). Cross-linking was carried out to stabilize the micelles [50]. Micelles of four different geometrical sizes in the range 13 to 40 nm, and hydrodynamic sizes in the range 47 to 142 nm, respectively, were prepared. Their labelling by [¹⁸F] was efficiently achieved, offering perspectives for PET imaging, albeit no in vitro nor in vivo study was reported.

<figure><img src="image_10.png" /><figcaption>**Figure 7.** Polynorbornene imide-based macromolecules.</figcaption></figure>

### 2.1.3. Dendrimers and Hyperbranched Molecules

Dendrimers are hyperbranched polymers that grow concentrically from a multifunctional core. Each starting branch corresponds to a dendron, itself ramified into new branches (Figure 8).
<figure><img src="image_11.png" /><figcaption>**Figure 8.** Schematic view of a dendrimer (from the core to the periphery the dashed circles represent 1st, 2nd, and 3rd generations).</figcaption></figure>

Dendrimers display a well-defined molecular architecture with many available func-
tional groups located either in their core, along the branches or at the periphery of the
different branches, and geometrical and hydrodynamic sizes that directly depend on the
generation number (a generation corresponding to the number of iterations of the poly-
merization). It allows a fine tuning of their solubility and stability in biological medium,
biocompatibility, pharmacokinetics, and facile functionalization with targeting ligands,
and/or radioelements. They find many applications in nanomedicine as drugs or drug
carriers [51].

Radiolabelling of dendrimers was the subject of a review in 2017 [52]. [¹⁸F]-radiolabelling of three generations of PAMAM (poly(amido amine)) dendrimers grown from a disulfide core was first reported. Conjugation to biotin released the half dendrimers (dendrons), each consisting of 4, 8, or 16 branches, with pendant amino groups that were further derivatized to expose arylboronic groups ready for radiolabelling as aryltrifluoroborates [53]. The impact of the size of the dendron on the biotin/avidin recognition was studied in vitro. PAMAM-NH₂ dendrimers of sixth generation were later radiolabelled [54] by electrostatic interaction. More recently, it was observed that a hydroxyl-terminated PAMAM dendrimer made of repetitive branching units of methyl acrylate and ethylene diamine, with a molecular weight of ~15,000 Da, was selectively taken up by reactive macrophages/microglia. Its radiolabelling allowed to evaluate its ability to detect innate immune activation in mice following lipopolysaccharide challenge [55]. This family of dendrimers is currently under clinical trial for the study of neuroinflammatory diseases in humans [56].

Interestingly, dendrimers have been used to stabilize inorganic nanoparticles. Radiola-
belling of these more complex architectures has been recently reviewed [57]; however there
is no mention of [18F]-radiolabelling.

## 2.2. *Inorganic Nanoplatforms*

Like the organic nanoparticles described above, inorganic nanoparticles are radiola-
belled first to study their pharmacokinetics and biodistribution in vivo. They occasionally
serve as inert carriers of molecular drugs or radiotracers, thus modifying their pharmacoki-
netics and biodistribution. They can also bring complementary properties to molecular
radiotracers anchored on their surface or sequestered in their core. Inorganic nanoparticles
can be designed with a mixed core–shell structure. As reported below, the shell might also
be implemented to favour surface functionalization. Alternatively, it may be of interest
to stabilize the properties of the core (luminescence or magnetism) or to bring a comple-
mentary physico-chemical property and afford multimodal platforms (e.g., association
between a magnetic core and a luminescent shell). The specific physico-chemical properties
of inorganic nanoparticles associated with their ease of functionalization make them ideal
nanoplatforms to develop multimodality imaging or theranostic agents. The nanomaterials
of interest are hereafter categorized following the nature of the core material that governs
their synthetic processes. When available, the main characteristics of the physico-chemistry
of their surface are indicated.
<figure><img src="image_11.png" /><figcaption>**Figure 8.** Schematic view of a dendrimer (from the core to the periphery the dashed circles represent 1st, 2nd, and 3rd generations).</figcaption></figure>

Dendrimers display a well-defined molecular architecture with many available func-
tional groups located either in their core, along the branches or at the periphery of the
different branches, and geometrical and hydrodynamic sizes that directly depend on the
generation number (a generation corresponding to the number of iterations of the poly-
merization). It allows a fine tuning of their solubility and stability in biological medium,
biocompatibility, pharmacokinetics, and facile functionalization with targeting ligands,
and/or radioelements. They find many applications in nanomedicine as drugs or drug
carriers [51].

Radiolabelling of dendrimers was the subject of a review in 2017 [52]. [¹⁸F]-radiolabelling of three generations of PAMAM (poly(amido amine)) dendrimers grown from a disulfide core was first reported. Conjugation to biotin released the half dendrimers (dendrons), each consisting of 4, 8, or 16 branches, with pendant amino groups that were further derivatized to expose arylboronic groups ready for radiolabelling as aryltrifluoroborates [53]. The impact of the size of the dendron on the biotin/avidin recognition was studied in vitro. PAMAM-NH₂ dendrimers of sixth generation were later radiolabelled [54] by electrostatic interaction. More recently, it was observed that a hydroxyl-terminated PAMAM dendrimer made of repetitive branching units of methyl acrylate and ethylene diamine, with a molecular weight of ~15,000 Da, was selectively taken up by reactive macrophages/microglia. Its radiolabelling allowed to evaluate its ability to detect innate immune activation in mice following lipopolysaccharide challenge [55]. This family of dendrimers is currently under clinical trial for the study of neuroinflammatory diseases in humans [56].

Interestingly, dendrimers have been used to stabilize inorganic nanoparticles. Radiola-
belling of these more complex architectures has been recently reviewed [57]; however there
is no mention of [18F]-radiolabelling.

## 2.2. *Inorganic Nanoplatforms*

Like the organic nanoparticles described above, inorganic nanoparticles are radiola-
belled first to study their pharmacokinetics and biodistribution in vivo. They occasionally
serve as inert carriers of molecular drugs or radiotracers, thus modifying their pharmacoki-
netics and biodistribution. They can also bring complementary properties to molecular
radiotracers anchored on their surface or sequestered in their core. Inorganic nanoparticles
can be designed with a mixed core–shell structure. As reported below, the shell might also
be implemented to favour surface functionalization. Alternatively, it may be of interest
to stabilize the properties of the core (luminescence or magnetism) or to bring a comple-
mentary physico-chemical property and afford multimodal platforms (e.g., association
between a magnetic core and a luminescent shell). The specific physico-chemical properties
of inorganic nanoparticles associated with their ease of functionalization make them ideal
nanoplatforms to develop multimodality imaging or theranostic agents. The nanomaterials
of interest are hereafter categorized following the nature of the core material that governs
their synthetic processes. When available, the main characteristics of the physico-chemistry
of their surface are indicated.
### 2.2.1. Metal Fluorides

Metal fluoride nanoparticles are a first class of studied nanomaterials, particularly those of rare earths, and closely related elements such as yttrium, of the type $NaMF_4$ or $KMF_4$ (M = Y$^{3+}$, Gd$^{3+}$, Lu$^{3+}$), and $MF_3$ (M = Y$^{3+}$, Gd$^{3+}$), or even bismuth. Their [$^{18}$F]-radiolabelling is generally achieved by ion exchange (see Section 3.2.2).

Fluorine displays a strong affinity to rare earth elements, hence the solubility of these fluorine salts in aqueous phases is very low (e.g., the solubility constant of $YF_3$ is as low as $8.62 \times 10^{-21}$ (25 °C)) [58], which limits leakage of the heavy elements in vivo.

The exceptional optical and magnetic properties of rare earths, and their high X-ray absorption coefficients make them attractive for a wide range of biomedical applications both for imaging (fluorescence, MRI, and CT) [59] and light-driven therapies (PDT, light activated drug delivery, etc.) [60].

For example, owing to its high magnetic moment, $Gd^{3+}$ in the form of gadolinium complexes is a well-used contrast agent for T1-weighted MRI, making the $Gd^{3+}$/$[^{18}F]$ pair a commonly studied combination for multimodal MRI/PET imaging. Thus, $GdF_3$ nanoparticles stabilized by human serum albumin (HSA) were developed, with geometric diameter $2.7 \pm 0.4$ nm, hydrodynamic size of $3.78 \pm 0.8$ nm, and a $\zeta$ potential of $-21.00 \pm 1.20$ mV in agreement with the negative charge of HSA at neutral pH. The use of HSA presented the added advantage of providing a suitable hydrophilicity and biocompatibility to the $GdF_3$ core. Given their moderate relaxivity ($5.11 \text{ mM}^{-1} \cdot \text{s}^{-1}$), T1-weighted MRI images with significant contrast could only be obtained 24 h after injection, when enough nanoparticles were accumulated in the tumour. Thus, in vivo radiolabelling of these nanoparticles was mandatory to follow their biodistribution in the early hours after injection ($<3$ h) [61].

$YF_3$ was also studied given its interest as a $Ln^{3+}$ host matrix to develop up-conversion nanoparticles (UCNP, see Figure 9). Large 5 nm $YF_3$ nanocrystals were synthesized in water in the presence of citric acid. They self-assembled into aggregates, the size of which could be adjusted from 30–40 nm to 50–70 nm by fine-tuning the yttrium salt/citrate ratio, reaction temperature, and time. Introduction of a targeting ligand, such as folic acid, helped maintain their size in the 20–30 nm range. Finally, the negative surface charges allowed electrostatic immobilization of doxorubicin, affording nano-objects of total hydrodynamic size of 73 nm and large stability in serum. A good uptake in lymph nodes was reported in mice [62].

The physico-chemical properties of rare earth fluoride nanoparticles can be tuned when doping these host matrices with other $Ln^{3+}$ ions. Two classes of luminescent nanomaterials are distinguished: up-conversion nanoparticles (UCNPs), capable of absorbing light in the NIR region while emitting with a large anti-Stokes shift at much higher energy (e.g., $YF_3$ doped with a $Ln^{3+}$ ion such as with $Yb^{3+}$, $Tm^{3+}$, or $Ho^{3+}$, or $Er^{3+}$, $KGdF_4$ nanoparticles doped with $Yb^{3+}$ or $Tm^{3+}$ ions [62]), and down-conversion nanoparticles (DCNPs) emitting at lower energy in the NIR-II window (e.g., $KGdF_4$ nanoparticles doped with $Eu^{3+}$ ions [59]). The basic principles are exemplified in Figure 9 for $Er^{3+}$ ions.

Recent review articles provide detailed information on UCNPs [60,63] and DCNPs, [64] respectively, as well as on recent developments on nanomaterials exhibiting both properties [65]. These converting properties, associated with narrow emission bandwidths, long fluorescence lifetimes, and photostability, have a wide range of applications in fluorescence biomedical imaging or for the light activation of prodrugs in vivo [66]. This has prompted the study of their pharmacokinetics and biodistribution. It is noteworthy that tracking these nanoparticles in vivo cannot rely on their intrinsic fluorescent properties as depth of penetration at the emitted wavelengths in the tissues is low ($<2$ cm). This is especially true for nanoparticles with diameters below 10 nm, which is unfortunate as their pharmacokinetics is expected to be more appropriate for bioimaging [67]. Indeed, fluorescence yields decrease with size due to a higher ratio of surface defects and a more efficient quenching by the solvent. As explained above, despite their magnetic properties, monitoring their trafficking in vivo can be performed neither by MRI nor computed tomography (CT) despite their high absorption coefficients because of the low sensitivity of these methods.
2.2.1. Metal Fluorides

Metal fluoride nanoparticles are a first class of studied nanomaterials, particularly
those of rare earths, and closely related elements such as yttrium, of the type NaMF₄ or
KMF₄ (M = Y³⁺, Gd³⁺, Lu³⁺), and MF₃ (M = Y³⁺, Gd³⁺), or even bismuth. Their [¹⁸F]-
radiolabelling is generally achieved by ion exchange (see Section 3.2.2).

Fluorine displays a strong affinity to rare earth elements, hence the solubility of these
fluorine salts in aqueous phases is very low (e.g., the solubility constant of YF₃ is as low as
8.62 × 10⁻²¹ (25 °C)) [58], which limits leakage of the heavy elements in vivo.

The exceptional optical and magnetic properties of rare earths, and their high X-ray
absorption coefficients make them attractive for a wide range of biomedical applications
both for imaging (fluorescence, MRI, and CT) [59] and light-driven therapies (PDT, light
activated drug delivery, etc.) [60].

For example, owing to its high magnetic moment, Gd3+ in the form of gadolinium
complexes is a well-used contrast agent for T1-weighted MRI, making the Gd3+/[¹⁸F] pair
a commonly studied combination for multimodal MRI/PET imaging. Thus, GdF₃ nanopar-
ticles stabilized by human serum albumin (HSA) were developed, with geometric diameter
2.7 ± 0.4 nm, hydrodynamic size of 3.78 ± 0.8 nm, and a ζ potential of -21.00 ± 1.20 mV
in agreement with the negative charge of HSA at neutral pH. The use of HSA presented the
added advantage of providing a suitable hydrophilicity and biocompatibility to the GdF₃
core. Given their moderate relaxivity (5.11 mM⁻¹·s⁻¹), T1-weighted MRI images with
significant contrast could only be obtained 24 h after injection, when enough nanoparticles
were accumulated in the tumour. Thus, in vivo radiolabelling of these nanoparticles was
mandatory to follow their biodistribution in the early hours after injection (<3 h) [61].

YF₃ was also studied given its interest as a Ln³⁺ host matrix to develop up-conversion
nanoparticles (UCNP, see Figure 9). Large 5 nm YF₃ nanocrystals were synthesized in
water in the presence of citric acid. They self-assembled into aggregates, the size of which
could be adjusted from 30–40 nm to 50–70 nm by fine-tuning the yttrium salt/citrate ratio,
reaction temperature, and time. Introduction of a targeting ligand, such as folic acid, helped
maintain their size in the 20–30 nm range. Finally, the negative surface charges allowed
electrostatic immobilization of doxorubicin, affording nano-objects of total hydrodynamic
size of 73 nm and large stability in serum. A good uptake in lymph nodes was reported in
mice [62].

The physico-chemical properties of rare earth fluoride nanoparticles can be tuned when doping these host matrices with other Ln³⁺ ions. Two classes of luminescent nanomaterials are distinguished: up-conversion nanoparticles (UCNPs), capable of absorbing light in the NIR region while emitting with a large anti-Stokes shift at much higher energy (e.g., YF₃ doped with a Ln³⁺ ion such as with Yb³⁺, Tm³⁺, or Ho³⁺, or Er³⁺, KGdF₄ nanoparticles doped with Yb³⁺ or Tm³⁺ ions [62]), and down-conversion nanoparticles (DCNPs) emitting at lower energy in the NIR-II window (e.g., KGdF₄ nanoparticles doped with Eu³⁺ ions [59]). The basic principles are exemplified in Figure 9 for Er³⁺ ions.

Recent review articles provide detailed information on UCNPs [60,63] and DCNPs, [64]
respectively, as well as on recent developments on nanomaterials exhibiting both proper-
ties [65]. These converting properties, associated with narrow emission bandwidths, long
fluorescence lifetimes, and photostability, have a wide range of applications in fluorescence
biomedical imaging or for the light activation of prodrugs in vivo [66]. This has prompted
the study of their pharmacokinetics and biodistribution. It is noteworthy that tracking these
nanoparticles in vivo cannot rely on their intrinsic fluorescent properties as depth of pene-
tration at the emitted wavelengths in the tissues is low (<2 cm). This is especially true for
nanoparticles with diameters below 10 nm, which is unfortunate as their pharmacokinetics
is expected to be more appropriate for bioimaging [67]. Indeed, fluorescence yields decrease
with size due to a higher ratio of surface defects and a more efficient quenching by the
solvent. As explained above, despite their magnetic properties, monitoring their trafficking
in vivo can be performed neither by MRI nor computed tomography (CT) despite their
high absorption coefficients because of the low sensitivity of these methods.
<figure><img src="image_12.png" /><figcaption>**Figure 9.** Simplified energy-level diagram depicting the luminescence mechanism of Er³⁺ ion as a typical example.</figcaption></figure>

Labelling with [¹⁸F] to perform monitoring by PET imaging is then a good alternative to fluorescence, CT, and MRI imaging to investigate the fate of these nanoparticles in vivo [68].

Based on this approach, the biodistribution of 3.8 nm large [¹⁸F]-KGdF₄:Eu nanoparticles, stabilized by oleic acid and coated with polyacrylic acid for hydrophilicity (final hydrodynamic size of 30 nm and ζ potential close to -13 mV in neutral pH), were studied. Efficient uptake in tumours was demonstrated [59]. The low uptake of [¹⁸F]-fluoride in bones (below 2% ID/g) confirmed that defluorination of the nanoparticles occurred only marginally despite the small size of the nanoparticles that could favour ion exchanges. The same observation (bone uptake below 2% ID/g) was made when NaGdF₄:Yb,Er nanoparticles (geometrical size 10 nm, coated with citrate ions) were studied [66]. An even lower bone uptake was measured when these nanoparticles were coated with alendronic or nitrilotri(methylphosphonic) acid. Contrarily, a ca 10% ID/g in bones was observed for [¹⁸F]-NaYF₄:Yb,Er nanoparticles with a Gd³⁺-doped surface aiming at promoting T1-weighted MRI. These nanoparticles displayed a geometrical size of 20 nm, ζ potential of +18 mV, and hydrodynamic size of 25 nm due to their surface coating with acids like folic acid, oleic acid, and aminocaproic acid for specific targeted applications [69]. Large 20 nm NaY₀.₂Gd₀.₆Yb₀.₁₈Er₀.₀₂F₄ nanoparticles with citrate ions coating were also studied in order to develop a nanoprobe for multimodal PET/MR/UCL imaging, with the same result regarding bone uptake [70]. Oleic acid stabilized NaY₀.₇₉F₄Yb₀.₂Tm₀.₀₁ nanoparticles of comparable size, but with a coating of α-cyclodextrin improving hydrophilicity and physiological stability, also showed a similar bone uptake after 2 h of incubation [71]. All these data seem to indicate that radiochemical release may be related to the cation nature (Gd³⁺ versus Y³⁺) or nanoparticle size, rather than to the nanoparticle coating.

A bismuth oxyfluoride matrix was also used to develop UCNPs for multimodal imaging [72]. The [¹⁸F]-doped bismuth nanoprobe (Na₀.₂₀Bi₀.₈₀O₀.₃₅F₁.₉₁: 20% Yb, 0.5% Tm) displayed a geometrical size in the range 30–60 nm, ζ potential of ≈0 mV, and a hydrodynamic size around 150 nm. It showed interesting X-ray attenuation characteristics for CT imaging, a good [¹⁸F]-radiolabelling yield for PET imaging, and UC luminescence properties.
<figure><img src="image_12.png" /><figcaption>**Figure 9.** Simplified energy-level diagram depicting the luminescence mechanism of Er³⁺ ion as a typical example.</figcaption></figure>

Labelling with [¹⁸F] to perform monitoring by PET imaging is then a good alternative to fluorescence, CT, and MRI imaging to investigate the fate of these nanoparticles in vivo [68].

Based on this approach, the biodistribution of 3.8 nm large [¹⁸F]-KGdF₄:Eu nanoparticles, stabilized by oleic acid and coated with polyacrylic acid for hydrophilicity (final hydrodynamic size of 30 nm and ζ potential close to -13 mV in neutral pH), were studied. Efficient uptake in tumours was demonstrated [59]. The low uptake of [¹⁸F]-fluoride in bones (below 2% ID/g) confirmed that defluorination of the nanoparticles occurred only marginally despite the small size of the nanoparticles that could favour ion exchanges. The same observation (bone uptake below 2% ID/g) was made when NaGdF₄:Yb,Er nanoparticles (geometrical size 10 nm, coated with citrate ions) were studied [66]. An even lower bone uptake was measured when these nanoparticles were coated with alendronic or nitrilotri(methylphosphonic) acid. Contrarily, a ca 10% ID/g in bones was observed for [¹⁸F]-NaYF₄:Yb,Er nanoparticles with a Gd³⁺-doped surface aiming at promoting T1-weighted MRI. These nanoparticles displayed a geometrical size of 20 nm, ζ potential of +18 mV, and hydrodynamic size of 25 nm due to their surface coating with acids like folic acid, oleic acid, and aminocaproic acid for specific targeted applications [69]. Large 20 nm NaY<sub>0.2</sub>Gd<sub>0.6</sub>Yb<sub>0.18</sub>Er<sub>0.02</sub>F<sub>4</sub> nanoparticles with citrate ions coating were also studied in order to develop a nanoprobe for multimodal PET/MR/UCL imaging, with the same result regarding bone uptake [70]. Oleic acid stabilized NaY<sub>0.79</sub>F<sub>4</sub>Yb<sub>0.2</sub>Tm<sub>0.01</sub> nanoparticles of comparable size, but with a coating of α-cyclodextrin improving hydrophilicity and physiological stability, also showed a similar bone uptake after 2 h of incubation [71]. All these data seem to indicate that radiochemical release may be related to the cation nature (Gd³⁺ versus Y³⁺) or nanoparticle size, rather than to the nanoparticle coating.

A bismuth oxyfluoride matrix was also used to develop UCNPs for multimodal imaging [72]. The [¹⁸F]-doped bismuth nanoprobe (Na<sub>0.20</sub>Bi<sub>0.80</sub>O<sub>0.35</sub>F<sub>1.91</sub>: 20% Yb, 0.5% Tm) displayed a geometrical size in the range 30–60 nm, ζ potential of ≈0 mV, and a hydrodynamic size around 150 nm. It showed interesting X-ray attenuation characteristics for CT imaging, a good [¹⁸F]-radiolabelling yield for PET imaging, and UC luminescence properties.
### 2.2.2. Metal Oxides and Hydroxides
#### Magnetic Nanoparticles

Magnetic nanoparticles (MNPs) are often based on iron oxides, more scarcely on cobalt. Indeed, iron oxide MNPs are non-toxic and cost-effective, and display sufficient magnetization values for biomedical applications. They mainly act as contrast agents for T1- and T2-weighted MRI depending on their size; moreover, they also find applications in thermotherapy, for magnetically driven drug delivery, or to treat anaemia in adults. Here again, as the low sensitivity of MRI does not afford accurate detection, radiolabelling of MNPs is a valued strategy to assess their pharmacokinetics.

Carbohydrate-coated iron oxide nanoparticles have been developed as contrast agents for magnetic resonance imaging of the liver and lymph nodes, and to treat anaemia in adults. They are generally prepared by coprecipitation of $Fe^{2+}$ and $Fe^{3+}$ ions in the aqueous phase in the presence of the carbohydrate polymer. Monocrystalline iron oxide nanoparticles (3–5 nm geometrical size) coated with a cross-linked dextran/epichlorin hydrin layer (final hydrodynamic size *ca* 30 nm) are well-known platforms (referred to as CLIO for Cross-Linked Iron Oxide) that, once treated with ammonia, expose $NH_2$ groups ready for derivatization [73] (Figure 10). A first study reported that, once radiolabelled, their detection threshold in vivo by PET was lowered by a factor of 200 in comparison to MRI and 50 in comparison to fluorescence imaging [74]. They were later successfully used to evidence the interest in dual fluorescence-mediated tomography (FMT) and PET when a far-red fluorophore was grafted on the $[^{18}F]$-CLIO platform [75] and to detect macrophages in aortic aneurysms by combining PET and CT imaging modalities [76]. The biodistribution of $[^{18}F]$-radiolabelled CLIO nanoparticles (*ca* 29 ± 3 nm geometrical size) in healthy mice was monitored by micro-PET/CT imaging [77]. It was evidenced that the nanoparticles could be efficiently concentrated to a specific anatomical location by application of an external magnetic field and could also be used for magnetically assisted drug delivery.

<figure><img src="image_13.png" /><figcaption>**Figure 10.** Schematic representation of a CLIO nanoplaneform.</figcaption></figure>

$[^{18}F]$-labelled ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (geometrical size 6.5 ± 0.5 nm), coated by polyacrylic acid and polydopamine layers, were developed as dual-modality PET/MRI nanoprobes. Their derivatization with $NH_2-PEG-C≡CH$ led to nanoparticles with an average hydrodynamic diameter of 40 nm and $ζ$ potential of –21 mV. It allowed the introduction of the radiolabel, and of an arginine–glycine–aspartic acid (RGD) peptide for targeting by copper-catalyzed alkyne–azide cycloaddition (CuAAC). The final $[^{18}F]$-RGD@USPIO nanoparticles were injected into MDAMB-231-bearing mice showing a three times higher tumour uptake in comparison to the non-
### 2.2.2. Metal Oxides and Hydroxides
#### Magnetic Nanoparticles

Magnetic nanoparticles (MNPs) are often based on iron oxides, more scarcely on cobalt. Indeed, iron oxide MNPs are non-toxic and cost-effective, and display sufficient magnetization values for biomedical applications. They mainly act as contrast agents for T1- and T2-weighted MRI depending on their size; moreover, they also find applications in thermotherapy, for magnetically driven drug delivery, or to treat anaemia in adults. Here again, as the low sensitivity of MRI does not afford accurate detection, radiolabelling of MNPs is a valued strategy to assess their pharmacokinetics.

Carbohydrate-coated iron oxide nanoparticles have been developed as contrast agents for magnetic resonance imaging of the liver and lymph nodes, and to treat anaemia in adults. They are generally prepared by coprecipitation of $Fe^{2+}$ and $Fe^{3+}$ ions in the aqueous phase in the presence of the carbohydrate polymer. Monocrystalline iron oxide nanoparticles (3–5 nm geometrical size) coated with a cross-linked dextran/epichlorin hydrin layer (final hydrodynamic size *ca* 30 nm) are well-known platforms (referred to as CLIO for Cross-Linked Iron Oxide) that, once treated with ammonia, expose $NH_2$ groups ready for derivatization [73] (Figure 10). A first study reported that, once radiolabelled, their detection threshold in vivo by PET was lowered by a factor of 200 in comparison to MRI and 50 in comparison to fluorescence imaging [74]. They were later successfully used to evidence the interest in dual fluorescence-mediated tomography (FMT) and PET when a far-red fluorophore was grafted on the [$^{18}$F]-CLIO platform [75] and to detect macrophages in aortic aneurysms by combining PET and CT imaging modalities [76]. The biodistribution of [$^{18}$F]-radiolabelled CLIO nanoparticles (*ca* 29 ± 3 nm geometrical size) in healthy mice was monitored by micro-PET/CT imaging [77]. It was evidenced that the nanoparticles could be efficiently concentrated to a specific anatomical location by application of an external magnetic field and could also be used for magnetically assisted drug delivery.

<figure><img src="image_13.png" /><figcaption>**Figure 10.** Schematic representation of a CLIO nanoplaneform.</figcaption></figure>

[$^{18}$F]-labelled ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (geometrical size 6.5 ± 0.5 nm), coated by polyacrylic acid and polydopamine layers, were developed as dual-modality PET/MRI nanoprobes. Their derivatization with $NH_2$-PEG-C≡CH led to nanoparticles with an average hydrodynamic diameter of 40 nm and $ζ$ potential of –21 mV. It allowed the introduction of the radiolabel, and of an arginine–glycine–aspartic acid (RGD) peptide for targeting by copper-catalyzed alkyne–azide cycloaddition (CuAAC). The final [$^{18}$F]-RGD@USPIO nanoparticles were injected into MDAMB-231-bearing mice showing a three times higher tumour uptake in comparison to the non-
vectorized nanoparticles. They were successfully used to monitor in vivo the therapeutic effect of anti-angiogenesis in breast cancer by combining PET and MRI [78]. Dopamine-coated Fe₃O₄ nanoparticles, functionalized or not with folic acid for vectorization (geometrical size 6.8 nm, hydrodynamic size *ca* 170 nm, and *ζ* potential value *ca* + 21 mV) and γ-Fe₂O₃ nanoparticles (average geometrical size of 250 nm, *ζ* potential value -28 mV) coated by PEG₆₀₀₀ and dopamine were radiolabelled via derivatization of the NH₂ groups of the dopamine ligand. The cellular uptake in MCF7 breast tumour cells was studied by measuring the radioactivity level after 1 and 2 h of incubation in vitro. Here again the highest uptake was achieved with the folic acid-modified nanoparticles (e.g., 35% ID/g, 16% ID/g, and *ca* 2% ID/g for folic acid-modified Fe₃O₄ nanoparticles, folate-free Fe₃O₄ nanoparticles, and free [¹⁸F]-probe, respectively). These nanostructures showed a dual-modality capability (PET and T2-MRI) [79]. The pendant carboxylic acid functions of a polyacrylic acid layer, used to impart iron oxide nanoparticles (8 nm geometrical size) with high stability in aqueous medium in a large range of ionic strengths (up to 600 mM) and pH values (≥4.5), with constant hydrodynamic size of *ca* 30 nm and *ζ* potential between -47 and -25 mV, were also used for further derivatization and radiolabelling (see Section 3.3.2). PET imaging in mice showed a non-specific accumulation in liver and spleen where significant radioactivity still persisted 4 h post-injection (22.5 ± 1.6 and 9.2 ± 1.5% ID per g, respectively) [80].

MnFe₂O₄ and Fe₃O₄ nanoparticles produced in organic phase were further coated by an aluminium hydroxide layer to stabilize their magnetic properties, making them efficient MRI contrast agents [81]. Hydrodynamic sizes of 21 nm, and very high positive *ζ* potential values of +72 mV and +50 mV for MnFe₂O₄@Al(OH)₃ and Fe₃O₄@Al(OH)₃ nanoparticles were reported, respectively. These properties were influenced by both the thickness and the hardness of the Al(OH)₃ layer, which depend on the synthetic method. The Al(OH)₃ layer also imparts hydrophilicity to the nanoparticles, highly increasing their colloidal stability in water. It also reacted easily with phosphonate derivatives (e.g., bisphosphonate polyethyleneglycol) and fluoride ions allowing their efficient radiolabelling (reaching 97%). Together, these two properties provide a dual-modality contrast agent for MRI and PET imaging. However, [¹⁸F]-fluoride leaking was observed in vivo.

Fe₃O₄@Al(OH)₃ nanoaggregates *ca* 250 nm geometrical size, consisting in magnetite cores (average geometrical size of 7 nm) entrapped in a matrix of porous aluminium hydroxide were also reported [82]. Rapid uptake of [¹⁸F] by the Al(OH)₃ surface layer allowed their fast and easy radiolabelling.

These [¹⁸F]-Fe₃O₄@Al(OH)₃ nanoparticles were successfully used to label mouse mesenchymal stem cells and study their biodistribution in healthy C57Bl/6 mice by combining PET and MR imaging [83]. Comparison between labelled and non-labelled nanoparticles showed the interest of combining MRI and PET imaging in order to improve the visualization and quantification of the nanoparticles in cells. Magnetic cores with a luminescent NaYF₄ (Yb, Tm) shell were also developed and radiolabelled to afford multimodal nanomaterials associating MRI, PET, and fluorescence imaging. For example, core-shell nanoparticles with various cation dopants, notably Co₀.₁₆Fe₂.₈₄O₄@NaYF₄ (Yb, Er) and Fe₃O₄@NaYF₄ (Yb, Tm), stabilized by bisphosphonate polyethyleneglycol conjugates were prepared. They exhibited high transverse relaxivity for MRI, high affinity for [¹⁸F]-fluoride for PET imaging, and fluorescent emissions from 500 to 800 nm under excitation at 980 nm [84].

## Semi-Conducting Nanoparticles

This class of nanomaterials is widely used in many fields, such as electronics, sensors, catalysis, cosmetics, food additives, and medicine owing to their photoactivity and redox properties.

Titanium dioxide (TiO₂) nanoparticles have been radiolabelled to study their biodistribution in rats both after i.v. and oral administration. The fate of nanoparticles with geometrical size 7–8 nm and hydrodynamic size 12 nm was followed up to 8 h after ad-
vectorized nanoparticles. They were successfully used to monitor in vivo the therapeutic effect of anti-angiogenesis in breast cancer by combining PET and MRI [78]. Dopamine-coated Fe₃O₄ nanoparticles, functionalized or not with folic acid for vectorization (geometrical size 6.8 nm, hydrodynamic size *ca* 170 nm, and *ζ* potential value *ca* + 21 mV) and γ-Fe₂O₃ nanoparticles (average geometrical size of 250 nm, *ζ* potential value -28 mV) coated by PEG₆₀₀₀ and dopamine were radiolabelled via derivatization of the NH₂ groups of the dopamine ligand. The cellular uptake in MCF7 breast tumour cells was studied by measuring the radioactivity level after 1 and 2 h of incubation in vitro. Here again the highest uptake was achieved with the folic acid-modified nanoparticles (e.g., 35% ID/g, 16% ID/g, and *ca* 2% ID/g for folic acid-modified Fe₃O₄ nanoparticles, folate-free Fe₃O₄ nanoparticles, and free [¹⁸F]-probe, respectively). These nanostructures showed a dual-modality capability (PET and T2-MRI) [79]. The pendant carboxylic acid functions of a polyacrylic acid layer, used to impart iron oxide nanoparticles (8 nm geometrical size) with high stability in aqueous medium in a large range of ionic strengths (up to 600 mM) and pH values (≥4.5), with constant hydrodynamic size of *ca* 30 nm and *ζ* potential between -47 and -25 mV, were also used for further derivatization and radiolabelling (see Section 3.3.2). PET imaging in mice showed a non-specific accumulation in liver and spleen where significant radioactivity still persisted 4 h post-injection (22.5 ± 1.6 and 9.2 ± 1.5% ID per g, respectively) [80].

MnFe₂O₄ and Fe₃O₄ nanoparticles produced in organic phase were further coated by an aluminium hydroxide layer to stabilize their magnetic properties, making them efficient MRI contrast agents [81]. Hydrodynamic sizes of 21 nm, and very high positive *ζ* potential values of +72 mV and +50 mV for MnFe₂O₄@Al(OH)₃ and Fe₃O₄@Al(OH)₃ nanoparticles were reported, respectively. These properties were influenced by both the thickness and the hardness of the Al(OH)₃ layer, which depend on the synthetic method. The Al(OH)₃ layer also imparts hydrophilicity to the nanoparticles, highly increasing their colloidal stability in water. It also reacted easily with phosphonate derivatives (e.g., bisphosphonate polyethyleneglycol) and fluoride ions allowing their efficient radiolabelling (reaching 97%). Together, these two properties provide a dual-modality contrast agent for MRI and PET imaging. However, [¹⁸F]-fluoride leaking was observed in vivo.

Fe₃O₄@Al(OH)₃ nanoaggregates *ca* 250 nm geometrical size, consisting in magnetite cores (average geometrical size of 7 nm) entrapped in a matrix of porous aluminium hydroxide were also reported [82]. Rapid uptake of [¹⁸F] by the Al(OH)₃ surface layer allowed their fast and easy radiolabelling.

These [¹⁸F]-Fe₃O₄@Al(OH)₃ nanoparticles were successfully used to label mouse mesenchymal stem cells and study their biodistribution in healthy C57Bl/6 mice by combining PET and MR imaging [83]. Comparison between labelled and non-labelled nanoparticles showed the interest of combining MRI and PET imaging in order to improve the visualization and quantification of the nanoparticles in cells. Magnetic cores with a luminescent NaYF₄ (Yb, Tm) shell were also developed and radiolabelled to afford multimodal nanomaterials associating MRI, PET, and fluorescence imaging. For example, core-shell nanoparticles with various cation dopants, notably Co₀.₁₆Fe₂.₈₄O₄@NaYF₄ (Yb, Er) and Fe₃O₄@NaYF₄ (Yb, Tm), stabilized by bisphosphonate polyethyleneglycol conjugates were prepared. They exhibited high transverse relaxivity for MRI, high affinity for [¹⁸F]-fluoride for PET imaging, and fluorescent emissions from 500 to 800 nm under excitation at 980 nm [84].

## Semi-Conducting Nanoparticles

This class of nanomaterials is widely used in many fields, such as electronics, sensors, catalysis, cosmetics, food additives, and medicine owing to their photoactivity and redox properties.

Titanium dioxide (TiO₂) nanoparticles have been radiolabelled to study their biodistribution in rats both after i.v. and oral administration. The fate of nanoparticles with geometrical size 7–8 nm and hydrodynamic size 12 nm was followed up to 8 h after ad-
ministration to the animals by combined PET-CT imaging. Preferential accumulation in the liver and a urinary excretion path were reported. Long after i.v. injection, radioactivity could be detected in the bones suggesting [¹⁸F]-fluoride leakage [85]. After oral administration, the labelled nanoparticles stayed almost quantitatively in the upper gastrointestinal tract. However, a slow and progressive accumulation in other organs (mainly the small intestine, the bones, and the bladder) was also observed. It is noteworthy that only few studies deal with the biodistribution and pharmacokinetics of TiO₂ nanoparticles, despite the fact that they are now used in many applications owing to their photoactivity [86,87] and are produced industrially on a large scale.

Two kinds of commercially available zinc oxide (ZnO) nanoparticles [88], with indicated geometrical sizes of 20 and 100 nm, respectively, were traced after their oral administration to mice. For this purpose, the nanoparticles were incubated with 6-hexynoic acid. The alkyne group could further undergo a CuAAC with a [¹⁸F]-labelled azide. After surface modification, the hydrodynamic sizes of the nanoparticles were *ca* 25 nm and 79 nm, for the 20 and 100 nm nanoparticles, respectively. The study showed that the nanoscale ZnO particles were retained longer in the gastrointestinal track in comparison to the sub-micrometer ones. The nanoparticles were not detected in other organs.

Ceria (CeO₂) nanoparticles functionalized with 3-aminopropyltriethoxysilane (geometrical size 5.6 nm, ζ potential +18.5 mV) were radiolabelled by coupling the amino pendant groups with [¹⁸F]-SFB. A geometrical size of 5.6 nm is reported, and a ζ potential of +18.5 mV, in agreement with the presence of amino surface groups, partially protonated in water at neutral pH. After i.v. injection in rats, the nanoparticles accumulated mainly in the liver (6% ID/g), lung, spleen, and kidneys (*ca* 2% ID/g) where the radioactivity level stayed constant along the *ca* 2 h monitoring by PET. Interestingly, it was evidenced that the very high level of radioactivity in the bladder and urine was due to soluble metabolites of the nanoparticles, not to the nanoparticles themselves [89].

Efficient radiolabelling of Y₂O₃ nanosheets was also reported in the presence or not of F127 as a surfactant but these were not used for in vivo studies [58].

Miscellaneous

Naked alumina (Al₂O₃) nanoparticles (5.5 nm geometrical size, 9 nm hydrodynamic size) were used without any surface modification to demonstrate the possibility to perform a direct radiolabelling by a ¹⁸O(p,n)¹⁸F nuclear reaction (see Section 3.2.1 for details), and explore their in vivo biodistribution in rats [90]. Commercial alumina nanoparticles of various sizes (see Table 2) were also [¹⁸F]-labelled by the same method [91].

<table><caption>**Table 2.** Main characteristics of the alumina nanoparticles studied (NR: data not reported).</caption><thead><tr><td>Geometrical Size (nm)</td><td>Hydrodynamic Size (nm)</td><td>ζ Potential (mV)</td><td>Reference</td></tr></thead><tbody><tr><td>5.5</td><td>9</td><td>NR</td><td>[90]</td></tr><tr><td>15</td><td>29.2</td><td>+3.3</td><td>[91]</td></tr><tr><td>35</td><td>35.1</td><td>+20.9</td><td>[91]</td></tr><tr><td rowspan="2">187</td><td>266</td><td>+9.3</td><td>[91]</td></tr><tr><td>2528</td><td>-5.7</td><td>[91]</td></tr></tbody></table>

Aluminium hydroxide (Al(OH)₃) nanoparticles with hydrodynamic size 1211 (±64) nm (Alhydrogel™) have been radiolabelled by incubation with [¹⁸F]-fluoride [92]. Given the recognized biocompatibility of aluminium hydrogels (e.g., used as adjuvants in vaccines), this prompted the development of aluminium hydroxide shells to facilitate the labelling of magnetic nanoparticles (see Section 2.2.2, refs. [81,82]) for use in MRI. After intramuscular and sub-cutaneous administration of the [¹⁸F]-Al(OH)₃ nanoparticles to mice, it was observed that all radioactivity remained at the site of injection over the 4 h period of monitoring. Contrarily, i.v. injection was followed by fast uptake in the liver and spleen, and by renal excretion.
ministration to the animals by combined PET-CT imaging. Preferential accumulation in the liver and a urinary excretion path were reported. Long after i.v. injection, radioactivity could be detected in the bones suggesting [¹⁸F]-fluoride leakage [85]. After oral administration, the labelled nanoparticles stayed almost quantitatively in the upper gastrointestinal tract. However, a slow and progressive accumulation in other organs (mainly the small intestine, the bones, and the bladder) was also observed. It is noteworthy that only few studies deal with the biodistribution and pharmacokinetics of TiO₂ nanoparticles, despite the fact that they are now used in many applications owing to their photoactivity [86,87] and are produced industrially on a large scale.

Two kinds of commercially available zinc oxide (ZnO) nanoparticles [88], with indicated geometrical sizes of 20 and 100 nm, respectively, were traced after their oral administration to mice. For this purpose, the nanoparticles were incubated with 6-hexynoic acid. The alkyne group could further undergo a CuAAC with a [¹⁸F]-labelled azide. After surface modification, the hydrodynamic sizes of the nanoparticles were *ca* 25 nm and 79 nm, for the 20 and 100 nm nanoparticles, respectively. The study showed that the nanoscale ZnO particles were retained longer in the gastrointestinal track in comparison to the sub-micrometer ones. The nanoparticles were not detected in other organs.

Ceria (CeO₂) nanoparticles functionalized with 3-aminopropyltriethoxysilane (geometrical size 5.6 nm, ζ potential +18.5 mV) were radiolabelled by coupling the amino pendant groups with [¹⁸F]-SFB. A geometrical size of 5.6 nm is reported, and a ζ potential of +18.5 mV, in agreement with the presence of amino surface groups, partially protonated in water at neutral pH. After i.v. injection in rats, the nanoparticles accumulated mainly in the liver (6% ID/g), lung, spleen, and kidneys (*ca* 2% ID/g) where the radioactivity level stayed constant along the *ca* 2 h monitoring by PET. Interestingly, it was evidenced that the very high level of radioactivity in the bladder and urine was due to soluble metabolites of the nanoparticles, not to the nanoparticles themselves [89].

Efficient radiolabelling of Y₂O₃ nanosheets was also reported in the presence or not of F127 as a surfactant but these were not used for in vivo studies [58].

Miscellaneous

Naked alumina (Al₂O₃) nanoparticles (5.5 nm geometrical size, 9 nm hydrodynamic size) were used without any surface modification to demonstrate the possibility to perform a direct radiolabelling by a ¹⁸O(p,n)¹⁸F nuclear reaction (see Section 3.2.1 for details), and explore their in vivo biodistribution in rats [90]. Commercial alumina nanoparticles of various sizes (see Table 2) were also [¹⁸F]-labelled by the same method [91].

<table><caption>**Table 2.** Main characteristics of the alumina nanoparticles studied (NR: data not reported).</caption><thead><tr><td>Geometrical Size (nm)</td><td>Hydrodynamic Size (nm)</td><td>ζ Potential (mV)</td><td>Reference</td></tr></thead><tbody><tr><td>5.5</td><td>9</td><td>NR</td><td>[90]</td></tr><tr><td>15</td><td>29.2</td><td>+3.3</td><td>[91]</td></tr><tr><td>35</td><td>35.1</td><td>+20.9</td><td>[91]</td></tr><tr><td rowspan="2">187</td><td>266</td><td>+9.3</td><td>[91]</td></tr><tr><td>2528</td><td>-5.7</td><td>[91]</td></tr></tbody></table>

Aluminium hydroxide (Al(OH)₃) nanoparticles with hydrodynamic size 1211 (±64) nm (Alhydrogel™) have been radiolabelled by incubation with [¹⁸F]-fluoride [92]. Given the recognized biocompatibility of aluminium hydrogels (e.g., used as adjuvants in vaccines), this prompted the development of aluminium hydroxide shells to facilitate the labelling of magnetic nanoparticles (see Section 2.2.2, refs. [81,82]) for use in MRI. After intramuscular and sub-cutaneous administration of the [¹⁸F]-Al(OH)₃ nanoparticles to mice, it was observed that all radioactivity remained at the site of injection over the 4 h period of monitoring. Contrarily, i.v. injection was followed by fast uptake in the liver and spleen, and by renal excretion.
Efficient radiolabelling of Y(OH)₃ and Gd(OH)₃ nanoparticles of large sizes and non-spherical shapes was also reported but these were not involved in any in vivo study [58].

Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, -HA-) nanoparticles of various sizes, shapes, and surface coatings (see Table 3) were radiolabelled by incubation with [¹⁸F]-fluoride [92]. In vivo study (in mice) showed different biodistributions of the nanoparticles depending on the administration route (i.v., intramuscular or sub-cutaneous) and aggregation state of the nanoparticles (the more aggregated being expectedly more trapped in the lungs). Interestingly, no radioactivity was observed in joints and bones, indicating that leakage of [¹⁸F]-fluoride was below detection limit. Similarly, HA layers grown on top of MnFe₂O₄@Al(OH)₃ and Fe₃O₄@Al(OH)₃ nanoparticles were radiolabelled [93]. Interestingly, the Al(OH)₃ layer of the template material was removed during the formation of the hydroxyapatite shell leading to MnFe₂O₄@HA and Fe₃O₄@HA nanoparticles with rod-like shapes (60.3 nm × 29.7 nm large for Fe₃O₄@HA, and almost twice as much for MnFe₂O₄@HA nanoparticles). The nanoparticles were stabilized by coordination of a bisphosphonate PEG-Me polymer on the HA layer leading to final hydrodynamic sizes of 50.7 nm and 60.3 nm, respectively. In this case, despite the presence of this surface ligand, high radiolabelling yields could be achieved.

<table><caption>**Table 3.** Main characteristics of the hydroxyapatite nanoparticles studied in [92] (NR: data not reported).</caption><thead><tr><th>Post-Synthetic Treatment</th><th>Geometrical Size (nm)</th><th>Shape</th><th>Hydrodynamic Size (nm)</th><th>ζ Potential (mV)</th></tr></thead><tbody><tr><td>Calcination</td><td>41.6 ± 1.8</td><td>Spherical nanocrystals/aggregated</td><td>2784 ± 158</td><td>-15.8 ± 0.2</td></tr><tr><td>Hydrothermal</td><td>107.5 ± 6.2 × 25.8 ± 0.9</td><td>Rod-like</td><td>829 ± 135</td><td>-6.0 ± 0.6</td></tr><tr><td>None</td><td>132.0 ± 30.3 × 12.6 ± 2.2</td><td>Needle-like</td><td>2046 ± 470 (main peak)</td><td>NR</td></tr><tr><td>Alendronate</td><td></td><td></td><td>169 ± 33 (main peak)</td><td>NR</td></tr><tr><td>MeO-PEG-COOH</td><td></td><td>Supposed to be identical to the above</td><td>214 ± 68</td><td>NR</td></tr></tbody></table>

### 2.2.3. Metal Sulfides, Selenides, and Tellurides

To the best of our knowledge, no metal sulfide nanoparticle has ever been radiolabelled by ¹⁸F. However, metal sulfide, especially ZnS, is present at the surface of many quantum dots (QDs), such as cadmium selenide or telluride nanoparticles. These well-known inorganic fluorescent nanoparticles find applications in many fields from optoelectronic devices [94] to bioimaging [95,96], owing to their brightness, sharp emission, low blinking, and long photostability. However, to preserve the excellent quantum yield of CdSe and CdTe nanocrystals, a shell of a semi-conducting material with a larger band gap should be present at the surface to confine the excited electrons within the core of the nanoparticle. ZnS has been largely used for this purpose.

Surprisingly, despite the huge amount of QDs produced and used in biomedicine since their discovery in the 1980s [96], few papers report their radiolabelling with ¹⁸F. One such example concerns commercially available, amine-modified, CdTe/ZnS nanoparticles (QD705, Quantum Dot Corp., Hayward, CA, USA) that were developed as potential dual-targeting, dual-modality imaging probes [97]. Thus, their surface was functionalized with a synthetic peptide combining an RGD motif (cyclo(RGDYK)) and a truncated bombesin sequence (7–14 BBN). This allowed the recognition of both integrin αvβ3, overexpressed in a variety of solid tumour types, and gastrin-releasing peptide receptors (GRPRs). Their hydrodynamic size was 123.5 nm. Radiolabelling with 4-nitrophenyl 2-[¹⁸F]fluoropropionate (see Section 3.3.2) provided a dual-targeted PET/NIRF imaging probe. Its efficiency was tested in mice. Clear delineation of the tumour site was possible by PET 30 min after injection, and by NIRF 1 h after injection.

CdSe/CdZnS nanoparticles (6–8 nm geometrical diameter) stabilized by modified phospholipids, exposing thiol end-groups for further derivatization and radiolabelling were also studied [16] (see Figure 11). This labelling strategy was used to investigate the dynamics of the in vivo biodistribution of non-targeted QDs. No renal clearance was observed, which is in agreement with their large diameter (hydrodynamic size after coating
Efficient radiolabelling of Y(OH)₃ and Gd(OH)₃ nanoparticles of large sizes and non-spherical shapes was also reported but these were not involved in any in vivo study [58].

Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, -HA-) nanoparticles of various sizes, shapes, and surface coatings (see Table 3) were radiolabelled by incubation with [¹⁸F]-fluoride [92]. In vivo study (in mice) showed different biodistributions of the nanoparticles depending on the administration route (i.v., intramuscular or sub-cutaneous) and aggregation state of the nanoparticles (the more aggregated being expectedly more trapped in the lungs). Interestingly, no radioactivity was observed in joints and bones, indicating that leakage of [¹⁸F]-fluoride was below detection limit. Similarly, HA layers grown on top of MnFe₂O₄@Al(OH)₃ and Fe₃O₄@Al(OH)₃ nanoparticles were radiolabelled [93]. Interestingly, the Al(OH)₃ layer of the template material was removed during the formation of the hydroxyapatite shell leading to MnFe₂O₄@HA and Fe₃O₄@HA nanoparticles with rod-like shapes (60.3 nm × 29.7 nm large for Fe₃O₄@HA, and almost twice as much for MnFe₂O₄@HA nanoparticles). The nanoparticles were stabilized by coordination of a bisphosphonate PEG-Me polymer on the HA layer leading to final hydrodynamic sizes of 50.7 nm and 60.3 nm, respectively. In this case, despite the presence of this surface ligand, high radiolabelling yields could be achieved.

<table><caption>**Table 3.** Main characteristics of the hydroxyapatite nanoparticles studied in [92] (NR: data not reported).</caption><thead><tr><th>Post-Synthetic Treatment</th><th>Geometrical Size (nm)</th><th>Shape</th><th>Hydrodynamic Size (nm)</th><th>ζ Potential (mV)</th></tr></thead><tbody><tr><td>Calcination</td><td>41.6 ± 1.8</td><td>Spherical nanocrystals/aggregated</td><td>2784 ± 158</td><td>-15.8 ± 0.2</td></tr><tr><td>Hydrothermal</td><td>107.5 ± 6.2 × 25.8 ± 0.9</td><td>Rod-like</td><td>829 ± 135</td><td>-6.0 ± 0.6</td></tr><tr><td>None</td><td>132.0 ± 30.3 × 12.6 ± 2.2</td><td>Needle-like</td><td>2046 ± 470 (main peak)</td><td>NR</td></tr><tr><td>Alendronate</td><td></td><td></td><td>169 ± 33 (main peak)</td><td>NR</td></tr><tr><td>MeO-PEG-COOH</td><td></td><td>Supposed to be identical to the above</td><td>214 ± 68</td><td>NR</td></tr></tbody></table>

### 2.2.3. Metal Sulfides, Selenides, and Tellurides

To the best of our knowledge, no metal sulfide nanoparticle has ever been radiolabelled by ¹⁸F. However, metal sulfide, especially ZnS, is present at the surface of many quantum dots (QDs), such as cadmium selenide or telluride nanoparticles. These well-known inorganic fluorescent nanoparticles find applications in many fields from optoelectronic devices [94] to bioimaging [95,96], owing to their brightness, sharp emission, low blinking, and long photostability. However, to preserve the excellent quantum yield of CdSe and CdTe nanocrystals, a shell of a semi-conducting material with a larger band gap should be present at the surface to confine the excited electrons within the core of the nanoparticle. ZnS has been largely used for this purpose.

Surprisingly, despite the huge amount of QDs produced and used in biomedicine since their discovery in the 1980s [96], few papers report their radiolabelling with ¹⁸F. One such example concerns commercially available, amine-modified, CdTe/ZnS nanoparticles (QD705, Quantum Dot Corp., Hayward, CA, USA) that were developed as potential dual-targeting, dual-modality imaging probes [97]. Thus, their surface was functionalized with a synthetic peptide combining an RGD motif (cyclo(RGDYK)) and a truncated bombesin sequence (7–14 BBN). This allowed the recognition of both integrin αvβ3, overexpressed in a variety of solid tumour types, and gastrin-releasing peptide receptors (GRPRs). Their hydrodynamic size was 123.5 nm. Radiolabelling with 4-nitrophenyl 2-[¹⁸F]fluoropropionate (see Section 3.3.2) provided a dual-targeted PET/NIRF imaging probe. Its efficiency was tested in mice. Clear delineation of the tumour site was possible by PET 30 min after injection, and by NIRF 1 h after injection.

CdSe/CdZnS nanoparticles (6–8 nm geometrical diameter) stabilized by modified phospholipids, exposing thiol end-groups for further derivatization and radiolabelling were also studied [16] (see Figure 11). This labelling strategy was used to investigate the dynamics of the in vivo biodistribution of non-targeted QDs. No renal clearance was observed, which is in agreement with their large diameter (hydrodynamic size after coating
> 20 nm). Interestingly, a few degradation products were observed, which were excreted after 90 min, while the QDs remained in the blood or accumulated in liver and spleen. This study evidenced a prolonged circulation in the blood, with a half-residence time in the order of 2 h.

<figure><img src="image_14.png" /><figcaption>**Figure 11.** [¹⁸F]-labelled PEG-phospholipid QD micelle [16].</figcaption></figure>

Presently, a wide range of QDs are commercially available with different pending functions allowing their conjugation with [¹⁸F]-prosthetic groups such as, e.g., [¹⁸F]-fluoroethyltosylate [98].

### 2.2.4. Zerovalent Metal Nanoparticles

Among zerovalent nanoparticles, gold nanoparticles have been the most studied for [¹⁸F]-radiolabelling. This is in line with the large range of applications offered by these nanoparticles in the biomedical field owing to their biocompatibility [99], ease of synthesis [100], and surface functionalization [101,102] associated with unique physico-chemical properties. Indeed, they display outstanding optical properties that can be tuned by adjusting their size and shape [103], large X-ray extinction coefficient [104], and potentially magnetic properties when their size and surface coating are properly adjusted [105]. Thus, they have been developed as colorimetric sensors [106], optical imaging agents [107,108] for phototherapy [109], and photoactivated antimicrobial applications [110,111]. However, their applications also encompass photoacoustic imaging [112], magnetic resonance imaging [105], radiotherapy (as radiosensitizers) [104,113], and theranostics [114,115]. They can also simply be used as drug delivery vehicles [116,117]. More recently, their promising effect on cell differentiation has also been acknowledged [118].

A first example of gold nanoparticles, [¹⁸F]-radiolabelling was achieved to study their biodistribution and in vivo pharmacokinetics. For example, 12 nm large gold nanoparticles designed for the treatment of Alzheimer's disease have been studied. These nanoparticles, prepared by the Turkevich method [119], were functionalized by a combination of CLPFFD and CK peptides that anchored strongly to the gold surface owing to thiol groups of the cysteine residue, while offering available amine functions for further conjugation with a [¹⁸F]-probe [120]. The final object displayed a hydrodynamic size of ca 23 nm and a ζ potential of -40 mV in plasma. A total of 2 h after injection in rats, only traces of
> 20 nm). Interestingly, a few degradation products were observed, which were excreted after 90 min, while the QDs remained in the blood or accumulated in liver and spleen. This study evidenced a prolonged circulation in the blood, with a half-residence time in the order of 2 h.

<figure><img src="image_14.png" /><figcaption>**Figure 11.** [¹⁸F]-labelled PEG-phospholipid QD micelle [16].</figcaption></figure>

Presently, a wide range of QDs are commercially available with different pending functions allowing their conjugation with [¹⁸F]-prosthetic groups such as, e.g., [¹⁸F]-fluoroethyltosylate [98].

### 2.2.4. Zerovalent Metal Nanoparticles

Among zerovalent nanoparticles, gold nanoparticles have been the most studied for [¹⁸F]-radiolabelling. This is in line with the large range of applications offered by these nanoparticles in the biomedical field owing to their biocompatibility [99], ease of synthesis [100], and surface functionalization [101,102] associated with unique physico-chemical properties. Indeed, they display outstanding optical properties that can be tuned by adjusting their size and shape [103], large X-ray extinction coefficient [104], and potentially magnetic properties when their size and surface coating are properly adjusted [105]. Thus, they have been developed as colorimetric sensors [106], optical imaging agents [107,108] for phototherapy [109], and photoactivated antimicrobial applications [110,111]. However, their applications also encompass photoacoustic imaging [112], magnetic resonance imaging [105], radiotherapy (as radiosensitizers) [104,113], and theranostics [114,115]. They can also simply be used as drug delivery vehicles [116,117]. More recently, their promising effect on cell differentiation has also been acknowledged [118].

A first example of gold nanoparticles, [¹⁸F]-radiolabelling was achieved to study their biodistribution and in vivo pharmacokinetics. For example, 12 nm large gold nanoparticles designed for the treatment of Alzheimer's disease have been studied. These nanoparticles, prepared by the Turkevich method [119], were functionalized by a combination of CLPFFD and CK peptides that anchored strongly to the gold surface owing to thiol groups of the cysteine residue, while offering available amine functions for further conjugation with a [¹⁸F]-probe [120]. The final object displayed a hydrodynamic size of ca 23 nm and a $\zeta$ potential of -40 mV in plasma. A total of 2 h after injection in rats, only traces of
the nanoparticles could be detected in the brain (<0.1% ID/g). Rather, a progressive accumulation in liver and spleen was observed and correlated to the size and negative surface charge of these nanoparticles. A biliary excretion path was suggested based on the radioactivity level measured in the intestine, but the highest radioactivity level was measured in the bladder and urine indicating that the main excretion path was urinary (supposing no detachment of the [¹⁸F]-probe from the nanoparticles). Radiolabelling of much smaller gold nanoparticles (3 nm geometrical size), prepared by a modified Brust-Schiffrin method [121], with a surface decorated by a combination of pegylated thiols to ensure their stability in aqueous phase and promote stealth, showed a slightly higher brain uptake (>0.13% ID/g 2 h after injection in rats) [122].

Radiolabelling was also envisaged to afford theranostic nanoplatforms. Gold nanoparticles with geometrical size between 15 and 40 nm (as determined by scanning electron microscopy) were produced by a modified Brust method [123] in the presence of [¹⁸F]-FDG-cysteamine adduct, which anchors to the gold nanoparticles surface via the thiol groups of the cysteamine. The OH groups of FDG were then used for conjugation with the anti-metadherin (anti-MTDH) antibody, specific for MTDH, a surface protein overexpressed in breast cancer cells. A slight decrease in ζ potential value from −13 mV for the gold-FDG nanoparticles to −18 mV for the final object is reported, showing an increased stability after conjugation with the targeting antibody. Injection of the gold nanoparticles in rats confirmed their potential for PET/CT imaging and their efficient targeting, prompting their development as radiosensitizers for the treatment of breast cancer.

Gold nanoparticles have also been chosen as representative objects to demonstrate the effectiveness of new labelling techniques [124]. Interestingly, Pd nanosheets have also been used for this purpose [125]. This is, to the best of our knowledge, the sole example of radiolabelling of zerovalent nanoparticles other than gold ones. These examples are discussed in Section 3.3.2.

### 2.2.5. C- and Si-Based Nanoparticles

Carbon- and silicon-based nanomaterials are attractive owing to their large availability, low cost, and limited toxicity. They display interesting properties for biomedical applications as depicted below.

*Carbon derivatives.* Carbon nanotubes, graphene, fullerenes, or nanodiamonds have been studied as potential platforms for theranostic applications [126], including delivery of radioisotopes for cancer therapy [127]. Being naturally hydrophobic and chemically rather inert, carbon-based nanomaterials are generally partially oxidized to afford surface OH, COOH, and/or epoxy groups to allow their functionalization (Figure 12).

<figure><img src="image_15.png" /><figcaption>**Figure 12.** Schematic representation of functional groups at the surface of nanodiamonds and exfoliated n-GO (not at scale).</figcaption></figure>
the nanoparticles could be detected in the brain (<0.1% ID/g). Rather, a progressive accumulation in liver and spleen was observed and correlated to the size and negative surface charge of these nanoparticles. A biliary excretion path was suggested based on the radioactivity level measured in the intestine, but the highest radioactivity level was measured in the bladder and urine indicating that the main excretion path was urinary (supposing no detachment of the [¹⁸F]-probe from the nanoparticles). Radiolabelling of much smaller gold nanoparticles (3 nm geometrical size), prepared by a modified Brust-Schiffrin method [121], with a surface decorated by a combination of pegylated thiols to ensure their stability in aqueous phase and promote stealth, showed a slightly higher brain uptake (>0.13% ID/g 2 h after injection in rats) [122].

Radiolabelling was also envisaged to afford theranostic nanoplatforms. Gold nanoparticles with geometrical size between 15 and 40 nm (as determined by scanning electron microscopy) were produced by a modified Brust method [123] in the presence of [¹⁸F]-FDG-cysteamine adduct, which anchors to the gold nanoparticles surface via the thiol groups of the cysteamine. The OH groups of FDG were then used for conjugation with the anti-metadherin (anti-MTDH) antibody, specific for MTDH, a surface protein overexpressed in breast cancer cells. A slight decrease in ζ potential value from −13 mV for the gold-FDG nanoparticles to −18 mV for the final object is reported, showing an increased stability after conjugation with the targeting antibody. Injection of the gold nanoparticles in rats confirmed their potential for PET/CT imaging and their efficient targeting, prompting their development as radiosensitizers for the treatment of breast cancer.

Gold nanoparticles have also been chosen as representative objects to demonstrate the effectiveness of new labelling techniques [124]. Interestingly, Pd nanosheets have also been used for this purpose [125]. This is, to the best of our knowledge, the sole example of radiolabelling of zerovalent nanoparticles other than gold ones. These examples are discussed in Section 3.3.2.

### 2.2.5. C- and Si-Based Nanoparticles

Carbon- and silicon-based nanomaterials are attractive owing to their large availability, low cost, and limited toxicity. They display interesting properties for biomedical applications as depicted below.

*Carbon derivatives.* Carbon nanotubes, graphene, fullerenes, or nanodiamonds have been studied as potential platforms for theranostic applications [126], including delivery of radioisotopes for cancer therapy [127]. Being naturally hydrophobic and chemically rather inert, carbon-based nanomaterials are generally partially oxidized to afford surface OH, COOH, and/or epoxy groups to allow their functionalization (Figure 12).

<figure><img src="image_15.png" /><figcaption>**Figure 12.** Schematic representation of functional groups at the surface of nanodiamonds and exfoliated n-GO (not at scale).</figcaption></figure>
In the case of nanodiamonds, this oxidation step may also be important to remove
the sp² carbon derivatives, such as graphite, that may form during their synthesis and
in which the nanodiamonds may be embedded. The surface of the nanodiamonds can
then be easily modified [128], making them especially interesting for applications in fluo-
rescence and magnetic resonance imaging, as well as in drug delivery [129]. Concerning
radiofluorination, taking advantage of the exposed OH groups, 7 nm large (geometrical
size) nanodiamonds were reacted with ω-aminopropyltriethoxysilane to finally display
amino groups at their surface. The latter were further conjugated with [¹⁸F]-SFB. Partial
aggregation of the nanodiamonds into larger objects (hydrodynamic diameter 680 nm)
was observed. These aggregates mainly accumulated in the lungs, and less in liver and
spleen after i.v. injection in rats. The addition of 5% Tween80 or 5% PEG₈₀₀ surfactant
had no major effect on their biodistribution, but tended to increase their bioavailability in
blood. Contrarily, the smaller nanoparticles or aggregates that could be recovered after
filtration (0.45 µm cut-off) were not detected in lungs nor spleen, and only traces could be
detected in the liver [130]. This study confirmed the strong impact of the hydrodynamic
size of nano-objects on biodistribution, and points out the need for a careful design of
surface functionalization to control this parameter in physiological media. Nano-graphene
oxide (nGO) was also studied, with the idea of developing a new radiotracer platform [131].
Disk-like nGO platelets were first prepared by exfoliation. They displayed a 50 nm large
geometrical diameter in average, and a ζ potential of −70 mV due to the presence of
ionised carboxylic acid functions at their surface. These carboxylic acid functions were
then used to introduce PEG chains via amidic coupling with NH₂-PEG-NH₂ and ensure a
good dispersion in aqueous phases such as PBS and DMEM. Accordingly, the ζ potential
value increased (−48 mV). The [¹⁸F]-radiolabelled nanomaterial displayed uptakes of 14%
and 8% of the injected dose in MCF7 and CT26 cells after 2 h, respectively. The biodistri-
bution in subcutaneous tumour-bearing CT-26 mice showed a passive accumulation of
the pegylated radiotracer in the tumour attributed to EPR effect (Figure 13), but with a
low standardized uptake value (SUV) of 0.2109 after 1 h. In comparison, [¹⁸F]-fluoride and
[¹⁸F]-nGO displayed SUV values of 0 and 0.0912, respectively, pointing out once again the
positive role of PEG chains in increasing the blood bioavailability of the nanomaterials.
Thus, with proper surface tailoring, nGO sheets could be used for the early detection of
tumours in bioimaging or to assist in drug delivery.

<figure><img src="image_16.png" /><figcaption>**Figure 13.** Illustration of the passive accumulation of ¹⁸F-nGO-PEG in a tumour-bearing murine model, achieved by the extravasation of nanoparticles through the increased permeability of the tumour vasculature (EPR effect). Created with Biorender.</figcaption></figure>

An attempt to label active carbon was also briefly reported [58], but to the best of
our knowledge, no work reports the [¹⁸F]-radiolabelling of other important carbon-based
In the case of nanodiamonds, this oxidation step may also be important to remove
the sp² carbon derivatives, such as graphite, that may form during their synthesis and
in which the nanodiamonds may be embedded. The surface of the nanodiamonds can
then be easily modified [128], making them especially interesting for applications in fluo-
rescence and magnetic resonance imaging, as well as in drug delivery [129]. Concerning
radiofluorination, taking advantage of the exposed OH groups, 7 nm large (geometrical
size) nanodiamonds were reacted with ω-aminopropyltriethoxysilane to finally display
amino groups at their surface. The latter were further conjugated with [¹⁸F]-SFB. Partial
aggregation of the nanodiamonds into larger objects (hydrodynamic diameter 680 nm)
was observed. These aggregates mainly accumulated in the lungs, and less in liver and
spleen after i.v. injection in rats. The addition of 5% Tween80 or 5% PEG₈₀₀ surfactant
had no major effect on their biodistribution, but tended to increase their bioavailability in
blood. Contrarily, the smaller nanoparticles or aggregates that could be recovered after
filtration (0.45 µm cut-off) were not detected in lungs nor spleen, and only traces could be
detected in the liver [130]. This study confirmed the strong impact of the hydrodynamic
size of nano-objects on biodistribution, and points out the need for a careful design of
surface functionalization to control this parameter in physiological media. Nano-graphene
oxide (nGO) was also studied, with the idea of developing a new radiotracer platform [131].
Disk-like nGO platelets were first prepared by exfoliation. They displayed a 50 nm large
geometrical diameter in average, and a ζ potential of −70 mV due to the presence of
ionised carboxylic acid functions at their surface. These carboxylic acid functions were
then used to introduce PEG chains via amidic coupling with NH₂-PEG-NH₂ and ensure a
good dispersion in aqueous phases such as PBS and DMEM. Accordingly, the ζ potential
value increased (−48 mV). The [¹⁸F]-radiolabelled nanomaterial displayed uptakes of 14%
and 8% of the injected dose in MCF7 and CT26 cells after 2 h, respectively. The biodistri-
bution in subcutaneous tumour-bearing CT-26 mice showed a passive accumulation of
the pegylated radiotracer in the tumour attributed to EPR effect (Figure 13), but with a
low standardized uptake value (SUV) of 0.2109 after 1 h. In comparison, [¹⁸F]-fluoride and
[¹⁸F]-nGO displayed SUV values of 0 and 0.0912, respectively, pointing out once again the
positive role of PEG chains in increasing the blood bioavailability of the nanomaterials.
Thus, with proper surface tailoring, nGO sheets could be used for the early detection of
tumours in bioimaging or to assist in drug delivery.

<figure><img src="image_16.png" /><figcaption>**Figure 13.** Illustration of the passive accumulation of ¹⁸F-nGO-PEG in a tumour-bearing murine model, achieved by the extravasation of nanoparticles through the increased permeability of the tumour vasculature (EPR effect). Created with Biorender.</figcaption></figure>

An attempt to label active carbon was also briefly reported [58], but to the best of
our knowledge, no work reports the [¹⁸F]-radiolabelling of other important carbon-based
nanomaterials, such as fullerenes, carbon nano-onions, and carbon nanotubes, despite their growing interest in the biomedical field [132].

*Silicon derivatives.* Silicon nanoparticles also present interesting fluorescence properties with long-lived excited states, high brightness, and stability, which make them suitable for the design of fluorescence imaging probes. When porous, they are promising vehicles for the delivery of anti-cancer drugs [133]. To facilitate its functionalization, the silicon surface can be treated to provide a silicon oxide layer with reactive surface OH groups, or a silicon carbide layer with a variety of activable hydroxyl groups, or a silicon hydrocarbide layer with methyl and methylene surface groups (Figure 14) [134]. The main characteristics of the [¹⁸F]-radiolabelled nanomaterials studied are reported in Table 4.

<figure><img src="image_17.png" /><figcaption>**Figure 14.** Schematic representation of the chemical groups at the surface of silicon nanomaterials depending on the preparation method.</figcaption></figure>

<table><caption>**Table 4.** Main characteristics of the [¹⁸F]-radiolabelled nanomaterials (NR: data not reported).</caption><thead><tr><th>Material</th><th>Surface Coating/Stabilizer</th><th>Geometrical Size</th><th>Specific Surface Area (m²/g)</th><th>Pore Size (nm)</th><th>Pore Volume (cm³/g)</th><th>Hydrodynamic Size (nm)</th><th>ζ Potential (mV)</th><th>Ref.</th></tr></thead><tbody><tr><td>TOPSi</td><td>None</td><td>1–10 μm</td><td>311</td><td>13</td><td>1.04</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>TCPSi</td><td>None</td><td>1–10 μm</td><td>262</td><td>16.8</td><td>1.10</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>THCPSi</td><td>None</td><td>1–10 μm</td><td>260</td><td>16.3</td><td>1.10</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>THCPSi</td><td>Fluorescein Isothiocyanate/polyethylene glycol-15-hydroxystearate</td><td>142 nm</td><td>202</td><td>9</td><td>0.51</td><td>NR</td><td>-30</td><td>[136]</td></tr><tr><td>THCPSi</td><td>HFBII Glyceryl monostearate + L-α-phosphatidyl choline</td><td>190 (±40) nm</td><td>NR</td><td>NR</td><td>NR</td><td>234</td><td>-21</td><td>[137]</td></tr><tr><td>HCPSi</td><td>PEG₁₂ chains with trans-cyclooctene end groups</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>193 (±30)</td><td>-40</td><td>[17]</td></tr><tr><td>THCPSi</td><td>None</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>165</td><td>-40</td><td>[138]</td></tr></tbody></table>

Porous silicon nanoparticles with SiOx (TOPSi), SiCx (TCPSi), and SiCxHy (THCPSi) surfaces have been compared [135]. After radiolabelling by direct incubation with [¹⁸F]-fluoride (see details in Section 3.2.2), it was demonstrated that only [¹⁸F]-THCPSi and [¹⁸F]-TCPSi displayed sufficient plasma stability (of the radiolabelling) for administration. Finally, only [¹⁸F]-THCPSi nanoparticles were studied in vivo. Hence, THCPSi nanoparticles of 142 nm geometrical sizes were assessed as potential carriers for oral drug delivery [136]. Indeed, the average pore diameter of the particles (9.0 nm), high specific surface area (202 m²/g), and the pore volume (0.51 cm³/g) are compatible with drug delivery applications. Their surface was modified with fluorescein isothiocyanate for in vitro studies and [¹⁸F]-labelled to allow in vivo biodistribution assessment in rats. As THCPSi nanoparticles tend to aggregate in aqueous media (especially in plasma), dispersion of the nanoparticles was ensured by addition of polyethyleneglycol-15-hydroxystearate surfactant. The ζ potential value was close to -30 mV. Oral administration to rats showed that the nanoparticles
nanomaterials, such as fullerenes, carbon nano-onions, and carbon nanotubes, despite their growing interest in the biomedical field [132].

*Silicon derivatives.* Silicon nanoparticles also present interesting fluorescence properties with long-lived excited states, high brightness, and stability, which make them suitable for the design of fluorescence imaging probes. When porous, they are promising vehicles for the delivery of anti-cancer drugs [133]. To facilitate its functionalization, the silicon surface can be treated to provide a silicon oxide layer with reactive surface OH groups, or a silicon carbide layer with a variety of activable hydroxyl groups, or a silicon hydrocarbide layer with methyl and methylene surface groups (Figure 14) [134]. The main characteristics of the [¹⁸F]-radiolabelled nanomaterials studied are reported in Table 4.

<figure><img src="image_17.png" /><figcaption>**Figure 14.** Schematic representation of the chemical groups at the surface of silicon nanomaterials depending on the preparation method.</figcaption></figure>

<table><caption>**Table 4.** Main characteristics of the [¹⁸F]-radiolabelled nanomaterials (NR: data not reported).</caption><thead><tr><th>Material</th><th>Surface Coating/Stabilizer</th><th>Geometrical Size</th><th>Specific Surface Area (m²/g)</th><th>Pore Size (nm)</th><th>Pore Volume (cm³/g)</th><th>Hydrodynamic Size (nm)</th><th>ζ Potential (mV)</th><th>Ref.</th></tr></thead><tbody><tr><td>TOPSi</td><td>None</td><td>1–10 µm</td><td>311</td><td>13</td><td>1.04</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>TCPSi</td><td>None</td><td>1–10 µm</td><td>262</td><td>16.8</td><td>1.10</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>THCPSi</td><td>None</td><td>1–10 µm</td><td>260</td><td>16.3</td><td>1.10</td><td>NR</td><td></td><td>[135]</td></tr><tr><td>THCPSi</td><td>Fluorescein Isothiocyanate/polyethylene glycol-15-hydroxystearate</td><td>142 nm</td><td>202</td><td>9</td><td>0.51</td><td>NR</td><td>-30</td><td>[136]</td></tr><tr><td>THCPSi</td><td>HFBII Glyceryl monostearate + L-α-phosphatidyl choline</td><td>190 (±40) nm</td><td>NR</td><td>NR</td><td>NR</td><td>234</td><td>-21</td><td>[137]</td></tr><tr><td>HCPSi</td><td>PEG<sub>12</sub> chains with trans-cyclooctene end groups</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>193 (±30)</td><td>-40</td><td>[17]</td></tr><tr><td>THCPSi</td><td></td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>165</td><td>-40</td><td>[138]</td></tr></tbody></table>

Porous silicon nanoparticles with SiOx (TOPSi), SiCx (TCPSi), and SiCxHy (THCPSi) surfaces have been compared [135]. After radiolabelling by direct incubation with [¹⁸F]-fluoride (see details in Section 3.2.2), it was demonstrated that only [¹⁸F]-THCPSi and [¹⁸F]-TCPSi displayed sufficient plasma stability (of the radiolabelling) for administration. Finally, only [¹⁸F]-THCPSi nanoparticles were studied in vivo. Hence, THCPSi nanoparticles of 142 nm geometrical sizes were assessed as potential carriers for oral drug delivery [136]. Indeed, the average pore diameter of the particles (9.0 nm), high specific surface area (202 m²/g), and the pore volume (0.51 cm³/g) are compatible with drug delivery applications. Their surface was modified with fluorescein isothiocyanate for in vitro studies and [¹⁸F]-labelled to allow in vivo biodistribution assessment in rats. As THCPSi nanoparticles tend to aggregate in aqueous media (especially in plasma), dispersion of the nanoparticles was ensured by addition of polyethyleneglycol-15-hydroxystearate surfactant. The ζ potential value was close to -30 mV. Oral administration to rats showed that the nanoparticles
did not cross the intestinal wall, as only a negligible amount of radioactivity was detected in the blood or organs outside the gastrointestinal tract. Moreover, i.v. injection led to accumulation of the nanoparticles in spleen and liver (ca 14% and 7% ID/g after 30 min, respectively). THCPSi nanoparticles with higher specific surface area (323 (±3) m²/g) but lower pore volume (0.48 (±0.01) cm³/g) and pore diameter (7.4 (±0.2) nm) were also studied [137]. After radiolabelling, these nanoparticles were coated by self-assembly with a fungal protein leading to very stable suspensions both in PBS and plasma. Upon coating the hydrodynamic size increased from 215 to 324 nm and the average ζ potential increased from -33 to -21 mV. After i.v. injection to rats, the highest uptake of the nanoparticles was seen in the liver and spleen (ca 10 and 9% ID/g after 30 min, respectively), and in the lungs (ca 2% ID/g after 30 min). Less lung uptake was observed in comparison with uncoated THCPSi nanoparticles, indicating that the protein coating efficiently prevented in vivo aggregation of the nanoparticles. It was also noted that this coating prevented [¹⁸F]-fluoride leakage as no radioactivity could be detected in the bones. Similarly, THCPSi nanoparticles encapsulated into solid lipids frequently used as nano-carriers (glyceryl monostearate and L-α-phosphatidyl choline) were investigated [17]. They displayed a large geometrical size distribution centred on 190 (±40) nm. After coating, the hydrodynamic size was 193 (±30) nm and the average ζ potential -40 mV. When intravenously injected to tumour-bearing mice, the nanoparticles accumulated mainly in the liver and spleen (ca 70 and 40% ID/g after 30 min, respectively), precluding their efficiency as drug delivery vehicles based on the EPR effect.

The biodistribution of THCPSi nanoparticles in rats was also studied according to a pre-targeting methodology, were radiolabelling occurs in vivo. For this purpose, THCPSi nanoparticles were surface modified with PEG chains bearing terminal *trans*-cyclooctene functional groups following the strategy outlined in Figure 15. Finally, a treatment with silver nitrate was performed to increase their stability in aqueous phases. Their fast and efficient reaction with a [¹⁸F]-radiotracer **16** bearing a tetrazine function confirmed the accumulation of the nanoparticles in liver and spleen.

*Silica nanoparticles*. Well-defined silica nanomaterials are present in many fields such as agriculture, environmental remediation, and the food industry given their low toxicity. They are also well used in medicine [139] owing to their ease of synthesis and functionalization [140,141]. Mesoporous silica nanoparticles (MSNPs) are particularly interesting as cargo nanomaterials [142].

Nanostructured hydrophilic fumed silica (Aerosil®200) was surface-functionalized with 3-aminopropyl-triethoxysilane to impart solubility in the aqueous phase and allow coupling with [¹⁸F]-SFB. It is noteworthy that fumed silica generally consists in chain-like assemblies of nanoparticles. The dispersion of the nanoparticles improved upon functionalization as the specific surface area increased from 200 m²/g to 325 m²/g. Nevertheless, the radiolabelled nanomaterial still displayed a broad distribution of geometrical sizes (from 30 to 130 nm). The hydrodynamic size was ca 400 (±80) nm and the ζ potential value -21 (±5) mV at neutral pH. After i.v. injection in mice, these nanoparticles accumulated in the spleen and lungs, but mainly in the liver, in agreement with their poor stability, large size distribution, and poor surface functionalization that favoured size exclusion trapping in lungs and their fast recognition by the reticuloendothelial system [143].

More promising, MSNPs with geometrical size in the range 100–150 nm, and displaying regular arrays of pores, were also radiolabelled to study their specific accumulation in solid tumours via the EPR effect, following a pre-targeting strategy [144]. The surface of these nanoparticles was covered by PEG₁₂ and PEG₂₄ chains to impart stability and stealth, and a sufficient blood circulation time. The end of the PEG₂₄ chain was equipped with a dibenzocyclooctyne (DBCO) group, for coupling in vivo with a [¹⁸F]-labelled alkylazide. A persistently strong tumour signal in the mice was observed in PET images when they were given DBCO-PEG-MSNs 24 h before injecting the [¹⁸F]-labelled molecular probe. In comparison, the non-pre-targeted mouse that received only the molecular probe had considerably less tumour uptake. This study clearly demonstrated the interest in MSNPs as
did not cross the intestinal wall, as only a negligible amount of radioactivity was detected in the blood or organs outside the gastrointestinal tract. Moreover, i.v. injection led to accumulation of the nanoparticles in spleen and liver (ca 14% and 7% ID/g after 30 min, respectively). THCPSi nanoparticles with higher specific surface area (323 (±3) m²/g) but lower pore volume (0.48 (±0.01) cm³/g) and pore diameter (7.4 (±0.2) nm) were also studied [137]. After radiolabelling, these nanoparticles were coated by self-assembly with a fungal protein leading to very stable suspensions both in PBS and plasma. Upon coating the hydrodynamic size increased from 215 to 324 nm and the average ζ potential increased from -33 to -21 mV. After i.v. injection to rats, the highest uptake of the nanoparticles was seen in the liver and spleen (ca 10 and 9% ID/g after 30 min, respectively), and in the lungs (ca 2% ID/g after 30 min). Less lung uptake was observed in comparison with uncoated THCPSi nanoparticles, indicating that the protein coating efficiently prevented in vivo aggregation of the nanoparticles. It was also noted that this coating prevented [¹⁸F]-fluoride leakage as no radioactivity could be detected in the bones. Similarly, THCPSi nanoparticles encapsulated into solid lipids frequently used as nano-carriers (glyceryl monostearate and L-α-phosphatidyl choline) were investigated [17]. They displayed a large geometrical size distribution centred on 190 (±40) nm. After coating, the hydrodynamic size was 193 (±30) nm and the average ζ potential -40 mV. When intravenously injected to tumour-bearing mice, the nanoparticles accumulated mainly in the liver and spleen (ca 70 and 40% ID/g after 30 min, respectively), precluding their efficiency as drug delivery vehicles based on the EPR effect.

The biodistribution of THCPSi nanoparticles in rats was also studied according to a pre-targeting methodology, were radiolabelling occurs in vivo. For this purpose, THCPSi nanoparticles were surface modified with PEG chains bearing terminal *trans*-cyclooctene functional groups following the strategy outlined in Figure 15. Finally, a treatment with silver nitrate was performed to increase their stability in aqueous phases. Their fast and efficient reaction with a [¹⁸F]-radiotracer **16** bearing a tetrazine function confirmed the accumulation of the nanoparticles in liver and spleen.

*Silica nanoparticles*. Well-defined silica nanomaterials are present in many fields such as agriculture, environmental remediation, and the food industry given their low toxicity. They are also well used in medicine [139] owing to their ease of synthesis and functionalization [140,141]. Mesoporous silica nanoparticles (MSNPs) are particularly interesting as cargo nanomaterials [142].

Nanostructured hydrophilic fumed silica (Aerosil®200) was surface-functionalized with 3-aminopropyl-triethoxysilane to impart solubility in the aqueous phase and allow coupling with [¹⁸F]-SFB. It is noteworthy that fumed silica generally consists in chain-like assemblies of nanoparticles. The dispersion of the nanoparticles improved upon functionalization as the specific surface area increased from 200 m²/g to 325 m²/g. Nevertheless, the radiolabelled nanomaterial still displayed a broad distribution of geometrical sizes (from 30 to 130 nm). The hydrodynamic size was ca 400 (±80) nm and the ζ potential value -21 (±5) mV at neutral pH. After i.v. injection in mice, these nanoparticles accumulated in the spleen and lungs, but mainly in the liver, in agreement with their poor stability, large size distribution, and poor surface functionalization that favoured size exclusion trapping in lungs and their fast recognition by the reticuloendothelial system [143].

More promising, MSNPs with geometrical size in the range 100–150 nm, and displaying regular arrays of pores, were also radiolabelled to study their specific accumulation in solid tumours via the EPR effect, following a pre-targeting strategy [144]. The surface of these nanoparticles was covered by PEG₁₂ and PEG₂₄ chains to impart stability and stealth, and a sufficient blood circulation time. The end of the PEG₂₄ chain was equipped with a dibenzocyclooctyne (DBCO) group, for coupling in vivo with a [¹⁸F]-labelled alkylazide. A persistently strong tumour signal in the mice was observed in PET images when they were given DBCO-PEG-MSNs 24 h before injecting the [¹⁸F]-labelled molecular probe. In comparison, the non-pre-targeted mouse that received only the molecular probe had considerably less tumour uptake. This study clearly demonstrated the interest in MSNPs as
a pre-targeting tool for the detection of solid tumours with radioelements of short half-life. It also suggests that this method could enable real-time monitoring of MSNPs during drug delivery.

<figure><img src="image_18.png" /><figcaption>**Figure 15.** (A) Preparation of TCO-THCPSi-nanoparticles and (B) description of the pre-targeted methodology developed in [138] for tracing the nanoparticles in healthy mice: TCO-THCPSi-nanoparticles were administered 15 min or 24 h before the i.v. injection of the [¹⁸F]-probe.</figcaption></figure>

Cylindrical silica nanoparticles doped with Gd³⁺ ions have been functionalized with [¹⁸F]-FDG to provide a multimodal imaging platform. The surface of these particles was conjugated with L-glutamine to improve their solubility and stability, resulting in a ζ potential of -29.3 mV, an average size of ca 130 nm, and a molecular weight of ca 3.3 kDa. These nanoparticles showed a high uptake in primary breast cancer and brain tumours that could be imaged by both PET and MRI [145].

Other silica nanoparticles with no specific surface engineering were also studied as control nanomaterials for the development of [¹⁸F]-radiolabelling methods of metal oxide and hydroxide nanoparticles [58,92].
a pre-targeting tool for the detection of solid tumours with radioelements of short half-life. It also suggests that this method could enable real-time monitoring of MSNPs during drug delivery.

<figure><img src="image_18.png" /><figcaption>**Figure 15.** (A) Preparation of TCO-THCPSi-nanoparticles and (B) description of the pre-targeted methodology developed in [138] for tracing the nanoparticles in healthy mice: TCO-THCPSi-nanoparticles were administered 15 min or 24 h before the i.v. injection of the [¹⁸F]-probe.</figcaption></figure>

Cylindrical silica nanoparticles doped with Gd³⁺ ions have been functionalized with [¹⁸F]-FDG to provide a multimodal imaging platform. The surface of these particles was conjugated with L-glutamine to improve their solubility and stability, resulting in a ζ potential of −29.3 mV, an average size of ca 130 nm, and a molecular weight of ca 3.3 kDa. These nanoparticles showed a high uptake in primary breast cancer and brain tumours that could be imaged by both PET and MRI [145].

Other silica nanoparticles with no specific surface engineering were also studied as control nanomaterials for the development of [¹⁸F]-radiolabelling methods of metal oxide and hydroxide nanoparticles [58,92].
**3. Methods for Generating Radiofluorinated Nanoparticles**

**3.1. Introduction to the Radiofluorination Methods**

As ¹⁸F cannot be found in nature, it has to be produced. This occurs using a cyclotron as particle accelerator (Figure 16). This generates high energy particles able to interact with the nucleus of atoms, promoting the nuclear reaction. For ¹⁸F production, two main approaches are commonly performed. The first one is based on the ²⁰Ne(d,α)¹⁸F reaction. The deuteron bombardment on a target filled with pressurized ²⁰Ne gas mixed with a few µmol of F₂ gas allows (after α emission) for the formation of a [¹⁸F]-fluorine atom, which provides ¹⁸F-¹⁹F diatomic molecules after atomic recombination. This source of electrophilic fluorine thus suffers isotopic dilution and low molar radioactivity.

<figure><img src="image_19.png" /><figcaption>**Figure 16.** Two modes of ¹⁸F production: electrophilic fluorine (**left**) vs. nucleophilic fluoride (**right**).</figcaption></figure>

A second method is based on the ¹⁸O(p,n)¹⁸F reaction. In this case, the proton bombardment of a target filled with [¹⁸O]-water provides (after neutron emission) [¹⁸F]-fluoride. This nucleophilic source of [¹⁸F]-fluorine is the obviously most stable chemical form of fluorine, commonly found in nature. It also exhibits significantly higher (at least three orders of magnitude higher) molar radioactivity. Thus [¹⁸F]-fluoride appears as the generally preferred source of radioactive, positron emitting, fluorine.

**3.1.1. Generation of [¹⁸F]-Fluoride from [¹⁸O]-H₂O**

As mentioned above, production of ¹⁸F, under its fluoride form, is carried out in a cyclotron by bombardment of high energy protons onto a target containing ¹⁸O isotopes. Although [¹⁸O]-H₂O is most commonly used, any material containing ¹⁸O might be involved in the nuclear transmutation. Still, it has to be pointed out that the interaction of the high energy particle beam with the target material involves a significant increase in temperature of the latter (above 1000 °C) [146], thus ruling out the direct involvement of sophisticated [¹⁸O]-labelled organic molecules. This explains why the ¹⁸O isotope is introduced via a water molecule. The bombardment thus provides fluoride in [¹⁸O]-water. Although this [¹⁸F]-fluoride could potentially be used directly, it should be noted that the process also generates cationic radioisotopes. Those are generally separated from the fluoride by way of an anion exchange column (Quaternary MethylAmine resin or QMA). The nature of these cationic pollutants obviously depends on the nature of the target material and can be characterized by gamma ray spectrum of the QMA [147]. [¹⁸F]-fluoride is then recovered from the QMA by elution with an aqueous solution, in which the composition sets the pH.

**3.1.2. Preparation of Fluoride for Nucleophilic Reaction in Organic Solvents**

To promote a nucleophilic reaction of [¹⁸F]-fluoride on an electrophilic organic partner, a careful preparation of the former is required. Indeed, nucleophilic reaction on organic substrates are hampered by the high hydration energy of fluoride. Moreover, hydrated fluoride, despite its negative charge, fully lacks any nucleophilicity in organic solvents. Thus, an appropriate preparation of the fluoride is not limited to the elimination of the cationic pollutants arising from the target but also requires the complete removal of water. This is usually achieved after elution of the QMA with a moderately basic mixture (CO₃²⁻ or HCO₃⁻) of water and acetonitrile containing either a tetraalkylammonium cation or a mixture of cryptand (classically Kryptofix® 222) and potassium salt. Once the radioactive fluoride has been eluted from the resin, an iteration of azeotropic distillations of the mixture is performed by heating under a moderate vacuum or a flow of inert gas with iterative addition of acetonitrile, providing the reactive dehydrated [¹⁸F]-fluoride, in which the
# 3. Methods for Generating Radiofluorinated Nanoparticles

## 3.1. Introduction to the Radiofluorination Methods

As ¹⁸F cannot be found in nature, it has to be produced. This occurs using a cyclotron as particle accelerator (Figure 16). This generates high energy particles able to interact with the nucleus of atoms, promoting the nuclear reaction. For ¹⁸F production, two main approaches are commonly performed. The first one is based on the ²⁰Ne(d,α)¹⁸F reaction. The deuteron bombardment on a target filled with pressurized ²⁰Ne gas mixed with a few µmol of F₂ gas allows (after α emission) for the formation of a [¹⁸F]-fluorine atom, which provides ¹⁸F-¹⁹F diatomic molecules after atomic recombination. This source of electrophilic fluorine thus suffers isotopic dilution and low molar radioactivity.

<figure><img src="image_19.png" /><figcaption>**Figure 16.** Two modes of ¹⁸F production: electrophilic fluorine (**left**) vs. nucleophilic fluoride (**right**).</figcaption></figure>

A second method is based on the ¹⁸O(p,n)¹⁸F reaction. In this case, the proton bombardment of a target filled with [¹⁸O]-water provides (after neutron emission) [¹⁸F]-fluoride. This nucleophilic source of [¹⁸F]-fluorine is the obviously most stable chemical form of fluorine, commonly found in nature. It also exhibits significantly higher (at least three orders of magnitude higher) molar radioactivity. Thus [¹⁸F]-fluoride appears as the generally preferred source of radioactive, positron emitting, fluorine.

### 3.1.1. Generation of [¹⁸F]-Fluoride from [¹⁸O]-H₂O

As mentioned above, production of ¹⁸F, under its fluoride form, is carried out in a cyclotron by bombardment of high energy protons onto a target containing ¹⁸O isotopes. Although [¹⁸O]-H₂O is most commonly used, any material containing ¹⁸O might be involved in the nuclear transmutation. Still, it has to be pointed out that the interaction of the high energy particle beam with the target material involves a significant increase in temperature of the latter (above 1000 °C) [146], thus ruling out the direct involvement of sophisticated [¹⁸O]-labelled organic molecules. This explains why the ¹⁸O isotope is introduced via a water molecule. The bombardment thus provides fluoride in [¹⁸O]-water. Although this [¹⁸F]-fluoride could potentially be used directly, it should be noted that the process also generates cationic radioisotopes. Those are generally separated from the fluoride by way of an anion exchange column (Quaternary MethylAmine resin or QMA). The nature of these cationic pollutants obviously depends on the nature of the target material and can be characterized by gamma ray spectrum of the QMA [147]. [¹⁸F]-fluoride is then recovered from the QMA by elution with an aqueous solution, in which the composition sets the pH.

### 3.1.2. Preparation of Fluoride for Nucleophilic Reaction in Organic Solvents

To promote a nucleophilic reaction of [¹⁸F]-fluoride on an electrophilic organic partner, a careful preparation of the former is required. Indeed, nucleophilic reaction on organic substrates are hampered by the high hydration energy of fluoride. Moreover, hydrated fluoride, despite its negative charge, fully lacks any nucleophilicity in organic solvents. Thus, an appropriate preparation of the fluoride is not limited to the elimination of the cationic pollutants arising from the target but also requires the complete removal of water. This is usually achieved after elution of the QMA with a moderately basic mixture (CO₃²⁻ or HCO₃⁻) of water and acetonitrile containing either a tetraalkylammonium cation or a mixture of cryptand (classically Kryptofix® 222) and potassium salt. Once the radioactive fluoride has been eluted from the resin, an iteration of azeotropic distillations of the mixture is performed by heating under a moderate vacuum or a flow of inert gas with iterative addition of acetonitrile, providing the reactive dehydrated [¹⁸F]-fluoride, in which the
solubility in the organic phase is enhanced by the cation (and its cryptand). Unless noted in the following description of the methods for radiolabelling nano-objects, this process is a common part of all the radiofluorination methods.

## 3.2. Direct Introduction of Fluoride through Interaction with an Internal Element of the Nanoparticle

### 3.2.1. Bombardment of a Nanoparticle Containing $^{18}$O

As mentioned previously, high energy proton bombardment on a target is deleterious for the structure of organic materials. Yet it has been shown that it is compatible with some inorganic nano-objects such as naked alumina nanoparticles [90]. Indeed, direct bombardment of $[^{18}O]-Al_2O_3$ allowed the formation of radiofluorinated alumina nanoparticles. Interestingly, as high energy proton bombardment promotes both $^{16}O(p, \alpha)^{13}N$ and $^{18}O(p,n)^{18}F$ reactions, both decay of $^{13}N$ and $^{18}F$ were observed. However, when carried out in $^{18}O$-enriched water with $[^{18}O]Al_2O_3$, the radioactive decay indicates the dominant presence of $^{18}F$ (71%). Furthermore, after 1 h, a complete decay of $^{13}N$ leaves $^{18}F$ as the sole radionuclide. TEM, XRD, and DLS analyses showed no evidence of a modification of the general morphology and structure of the nanoparticles. The same approach has also been performed on $^{18}O$-enriched titanium dioxide nanoparticles allowing the formation of $^{18}F$ containing $TiO_2$ nanoparticles [85]. High resolution gamma ray spectroscopy confirmed the generation of metal isotopes such as $^{48}V$ and $^{47}V$ and $^{44}Sc$ arising from interaction of low-incident protons with the stable isotopes of Ti (namely $^{47}Ti$ and $^{48}Ti$, of 7 and 74% natural abundance, respectively). A detailed analysis of all positron isotope emitters was also carried out allowing a precise determination of the relative abundance of all radionuclides. Longer-lived radionuclides are assumed to allow combination of in vivo and ex vivo studies (imaging and dissection/γ counting).

### 3.2.2. Direct Reaction of $[^{18}F]$-Fluoride with Elements of the Nanoparticle

A first convenient approach to introduce $[^{18}F]$-fluoride within a nanoparticle shall take advantage of the well-known affinity of numerous metal and main group elements for fluoride. Although simple in nature, this approach might be considered potentially deleterious for the physico-chemical properties of the nanoparticle, in which the integrity can hardly be assessed by conventional methods for radiation hazard reasons. Thus, characterizations shall either take advantage of analytical techniques available in radiochemical facilities or be carried out after radioactive decay. The molar amount of radioactive fluoride introduced is very low owing to its high specific activity, and the effect on the physico-chemical properties of the nanoparticle is expected to remain limited. Representative examples of this approach are provided below.

Direct radiofluorination of hydroxyapatite (HA), either untreated thermally, calcinated, or hydrothermally treated, and aluminium hydroxide nanoparticles with $^{18}F$ has been performed by incubation of the aqueous $[^{18}F]$-fluoride solution obtained from the cyclotron at room temperature [92]. The radiolabelling yield did not depend significantly on the preparation method but was impaired by the presence of alendronate or carboxylate-PEG-OMe at the surface. These ligands are indeed supposed to coordinate to surface calcium ions, forming calcium phosphonate or calcium carboxylate bonds, respectively, leaving less calcium ions available for labelling. After a detailed exploration of the reaction conditions, optimized conditions were found in which radiofluorination was shown to occur and reach a plateau after 5 min, exhibiting a high labelling efficiency even at concentrations of nanoparticle below 1 mg/mL. HA-based radiolabelled nanoparticles were also shown to be stable in human serum, whereas the aluminium hydroxide ones were prone to release the radioactivity. In a closely related approach from a practical point of view, radiofluorination of a range of rare earth-based up-converting nanoparticles (UCNPs) has been performed by incubation with 100 µL of an aqueous solution of $[^{18}F]$-fluoride at room temperature with sonication for 1 min [58,70]. Radiochemical conversion rates ranging from 50 to 90% have been reported, establishing the efficiency of the process. Interestingly, silica encapsulated
solubility in the organic phase is enhanced by the cation (and its cryptand). Unless noted in the following description of the methods for radiolabelling nano-objects, this process is a common part of all the radiofluorination methods.

## 3.2. Direct Introduction of Fluoride through Interaction with an Internal Element of the Nanoparticle

### 3.2.1. Bombardment of a Nanoparticle Containing $^{18}$O

As mentioned previously, high energy proton bombardment on a target is deleterious for the structure of organic materials. Yet it has been shown that it is compatible with some inorganic nano-objects such as naked alumina nanoparticles [90]. Indeed, direct bombardment of $[^{18}O]-Al_2O_3$ allowed the formation of radiofluorinated alumina nanoparticles. Interestingly, as high energy proton bombardment promotes both $^{16}O(p, \alpha)^{13}N$ and $^{18}O(p,n)^{18}F$ reactions, both decay of $^{13}N$ and $^{18}F$ were observed. However, when carried out in $^{18}O$-enriched water with $[^{18}O]Al_2O_3$, the radioactive decay indicates the dominant presence of $^{18}F$ (71%). Furthermore, after 1 h, a complete decay of $^{13}N$ leaves $^{18}F$ as the sole radionuclide. TEM, XRD, and DLS analyses showed no evidence of a modification of the general morphology and structure of the nanoparticles. The same approach has also been performed on $^{18}O$-enriched titanium dioxide nanoparticles allowing the formation of $^{18}F$ containing $TiO_2$ nanoparticles [85]. High resolution gamma ray spectroscopy confirmed the generation of metal isotopes such as $^{48}V$ and $^{47}V$ and $^{44}Sc$ arising from interaction of low-incident protons with the stable isotopes of Ti (namely $^{47}Ti$ and $^{48}Ti$, of 7 and 74% natural abundance, respectively). A detailed analysis of all positron isotope emitters was also carried out allowing a precise determination of the relative abundance of all radionuclides. Longer-lived radionuclides are assumed to allow combination of in vivo and ex vivo studies (imaging and dissection/γ counting).

### 3.2.2. Direct Reaction of $[^{18}F]$-Fluoride with Elements of the Nanoparticle

A first convenient approach to introduce $[^{18}F]$-fluoride within a nanoparticle shall take advantage of the well-known affinity of numerous metal and main group elements for fluoride. Although simple in nature, this approach might be considered potentially deleterious for the physico-chemical properties of the nanoparticle, in which the integrity can hardly be assessed by conventional methods for radiation hazard reasons. Thus, characterizations shall either take advantage of analytical techniques available in radiochemical facilities or be carried out after radioactive decay. The molar amount of radioactive fluoride introduced is very low owing to its high specific activity, and the effect on the physico-chemical properties of the nanoparticle is expected to remain limited. Representative examples of this approach are provided below.

Direct radiofluorination of hydroxyapatite (HA), either untreated thermally, calcinated, or hydrothermally treated, and aluminium hydroxide nanoparticles with $^{18}F$ has been performed by incubation of the aqueous $[^{18}F]$-fluoride solution obtained from the cyclotron at room temperature [92]. The radiolabelling yield did not depend significantly on the preparation method but was impaired by the presence of alendronate or carboxylate-PEG-OMe at the surface. These ligands are indeed supposed to coordinate to surface calcium ions, forming calcium phosphonate or calcium carboxylate bonds, respectively, leaving less calcium ions available for labelling. After a detailed exploration of the reaction conditions, optimized conditions were found in which radiofluorination was shown to occur and reach a plateau after 5 min, exhibiting a high labelling efficiency even at concentrations of nanoparticle below 1 mg/mL. HA-based radiolabelled nanoparticles were also shown to be stable in human serum, whereas the aluminium hydroxide ones were prone to release the radioactivity. In a closely related approach from a practical point of view, radiofluorination of a range of rare earth-based up-converting nanoparticles (UCNPs) has been performed by incubation with 100 µL of an aqueous solution of $[^{18}F]$-fluoride at room temperature with sonication for 1 min [58,70]. Radiochemical conversion rates ranging from 50 to 90% have been reported, establishing the efficiency of the process. Interestingly, silica encapsulated
particles, as well as SiO₂ and ZnO nanoparticles, were poorly to unreactive under the same conditions, indicating a non-expected screening effect of silica. This lack of reactivity in the radiofluorination conditions used indirectly confirmed the rare earth fluoride bonding in the UCNPs. Stability was assessed by incubation in PBS for 2 h, showing a variable dissociation of radioactive fluoride depending on the nature of the anion associated with the rare earth cation (fluoride and hydroxide showing the lowest leakage of [¹⁸F]-fluoride). This method was more recently applied to UCNPs capped with etidronic acid, alendronic acid, and nitrilotrimethylphosphonic acid, where the former exhibited a low propensity to radiofluorination (RCY < 10%) while the two others were equally efficiently radiolabelled (43%) [66]. The affinity of rare earth for fluoride was also the basis for the radiolabelling of YF₃ nanoparticles loaded with a range of surface ligands such as citric acid, folic acid, doxorubicin, or PEG linkage [62]. Again, a simple incubation (30 min) at room temperature provided excellent radiochemical yields ranging from 84 to 96%, exhibiting an excellent kinetic profile of the reaction. Interestingly, purified nanoparticles incubated for 30 min in human or mouse serum released only up to 6% of their radioactivity, exhibiting no further release upon prolonged incubation, and thus establishing the good stability of these constructs. This illustrates again a fast establishment of the equilibrium, but leaves open the question of further release of fluoride after injection in a living body. Related core–shell colloidal nanoparticles made of iron oxide surrounded by a NaYbF₄ or NaErF₄ shell have been radiolabelled by a simple isotope exchange occurring on incubation with aqueous fluoride at room temperature, under similar reaction conditions, albeit with lower efficiencies (38%) [84]. The stability of the radiolabelling was confirmed by prolonged incubation in human serum, centrifugation, and activity counting of the supernatant and the nanoparticles filtered. Over 85% of the radioactivity was shown to remain on the nanoparticle. An effect of the size of the nanoparticles was somehow observed. Indeed, Sub-5nm KGdF₄:Eu³⁺ nanoparticles coated with polyacrylic acid have been shown to undergo direct radiofluorination in aqueous solution via a simple 10 min incubation with [¹⁸F]-KF and removal of the unreacted fluoride by centrifugation [59]. The radiolabelling yield has been estimated to be around 50%, which is somehow lower than for larger (around 25 nm, hence exhibiting a higher surface) rare earth nanoparticles for which RCY reached 80%.

As previously illustrated, the impact of the coating on the radiofluorination of nanoparticles is an obvious matter that has been also illustrated by involving aluminium. Indeed, Fe₃O₄ nanoparticles with a layer of Al(OH)₃ exhibiting a hydrodynamic diameter of 573 nm and a ζ potential of +18 mV have been efficiently radiofluorinated through an incubation with a saline [¹⁸F]-fluoride solution for 10 min at room temperature [83].

Despite silica coating being shown to be somehow detrimental for radiolabelling UCNPs, silicon is well-known for its affinity to fluoride; thus, silicon-based nanoparticles are also illustrated in this section. Porous silicon nanoparticles either thermally hydrocarbonized (THCPSi), thermally oxidized (TOPSi), and thermally carbonized (TCPSi) have been directly radiofluorinated (generation of a Si–F bond) by reaction with pre-dried [¹⁸F]-fluoride, Kryptofix® 222, and potassium fluoride in various solvents at various temperatures [135]. This study indicates a moderate interest of adding acetic acid to perform the reaction, although it was previously reported as advantageous for the radiofluorination of Si–H-functionalized surfaces. A reaction carried out in DMF at 120 °C gave an RCY of up to 81% with 1 h of synthesis time. THCPSi nanoparticles similarly radiolabelled by incubation in the presence of acetic acid could be then further coated with functional proteins to modulate their biodistribution. The overall RCY after coating was lower, reaching only 40%, but coating allowed a formulation in saline aqueous solutions [137]. Coating of THCPSi with solid lipids allowed the formation of a solid lipid nanocomposite containing [¹⁸F]-THCPSi ([¹⁸F]-THCPSi-SLNCs) in up to 30% RCY [17]. Unsurprisingly, the extra coating occurred with a decrease in the molar radioactivity of the particles.

Finally, the direct radiolabelling of organic nano-objects has also been performed as illustrated for the specific case of nano-graphene oxide, more specifically nGO-PEG
particles, as well as SiO₂ and ZnO nanoparticles, were poorly to unreactive under the same conditions, indicating a non-expected screening effect of silica. This lack of reactivity in the radiofluorination conditions used indirectly confirmed the rare earth fluoride bonding in the UCNPs. Stability was assessed by incubation in PBS for 2 h, showing a variable dissociation of radioactive fluoride depending on the nature of the anion associated with the rare earth cation (fluoride and hydroxide showing the lowest leakage of [¹⁸F]-fluoride). This method was more recently applied to UCNPs capped with etidronic acid, alendronic acid, and nitrilotrimethylphosphonic acid, where the former exhibited a low propensity to radiofluorination (RCY < 10%) while the two others were equally efficiently radiolabelled (43%) [66]. The affinity of rare earth for fluoride was also the basis for the radiolabelling of YF₃ nanoparticles loaded with a range of surface ligands such as citric acid, folic acid, doxorubicin, or PEG linkage [62]. Again, a simple incubation (30 min) at room temperature provided excellent radiochemical yields ranging from 84 to 96%, exhibiting an excellent kinetic profile of the reaction. Interestingly, purified nanoparticles incubated for 30 min in human or mouse serum released only up to 6% of their radioactivity, exhibiting no further release upon prolonged incubation, and thus establishing the good stability of these constructs. This illustrates again a fast establishment of the equilibrium, but leaves open the question of further release of fluoride after injection in a living body. Related core–shell colloidal nanoparticles made of iron oxide surrounded by a NaYbF₄ or NaErF₄ shell have been radiolabelled by a simple isotope exchange occurring on incubation with aqueous fluoride at room temperature, under similar reaction conditions, albeit with lower efficiencies (38%) [84]. The stability of the radiolabelling was confirmed by prolonged incubation in human serum, centrifugation, and activity counting of the supernatant and the nanoparticles filtered. Over 85% of the radioactivity was shown to remain on the nanoparticle. An effect of the size of the nanoparticles was somehow observed. Indeed, Sub-5nm KGdF₄:Eu³⁺ nanoparticles coated with polyacrylic acid have been shown to undergo direct radiofluorination in aqueous solution via a simple 10 min incubation with [¹⁸F]-KF and removal of the unreacted fluoride by centrifugation [59]. The radiolabelling yield has been estimated to be around 50%, which is somehow lower than for larger (around 25 nm, hence exhibiting a higher surface) rare earth nanoparticles for which RCY reached 80%.

As previously illustrated, the impact of the coating on the radiofluorination of nanoparticles is an obvious matter that has been also illustrated by involving aluminium. Indeed, Fe₃O₄ nanoparticles with a layer of Al(OH)₃ exhibiting a hydrodynamic diameter of 573 nm and a ζ potential of +18 mV have been efficiently radiofluorinated through an incubation with a saline [¹⁸F]-fluoride solution for 10 min at room temperature [83].

Despite silica coating being shown to be somehow detrimental for radiolabelling UCNPs, silicon is well-known for its affinity to fluoride; thus, silicon-based nanoparticles are also illustrated in this section. Porous silicon nanoparticles either thermally hydrocarbonized (THCPSi), thermally oxidized (TOPSi), and thermally carbonized (TCPSi) have been directly radiofluorinated (generation of a Si–F bond) by reaction with pre-dried [¹⁸F]-fluoride, Kryptofix® 222, and potassium fluoride in various solvents at various temperatures [135]. This study indicates a moderate interest of adding acetic acid to perform the reaction, although it was previously reported as advantageous for the radiofluorination of Si–H-functionalized surfaces. A reaction carried out in DMF at 120 °C gave an RCY of up to 81% with 1 h of synthesis time. THCPSi nanoparticles similarly radiolabelled by incubation in the presence of acetic acid could be then further coated with functional proteins to modulate their biodistribution. The overall RCY after coating was lower, reaching only 40%, but coating allowed a formulation in saline aqueous solutions [137]. Coating of THCPSi with solid lipids allowed the formation of a solid lipid nanocomposite containing [¹⁸F]-THCPSi ([¹⁸F]-THCPSi-SLNCs) in up to 30% RCY [17]. Unsurprisingly, the extra coating occurred with a decrease in the molar radioactivity of the particles.

Finally, the direct radiolabelling of organic nano-objects has also been performed as illustrated for the specific case of nano-graphene oxide, more specifically nGO-PEG
derivatives [131]. With this substrate, the radiofluorination was shown to occur upon heating for 1 h in a TFA aqueous solution (pH 5.5) at 80 °C. The reaction was reported as highly efficient (RCY over 96%). FT-IR investigation of [¹⁹F]-fluorinated nGO indicates a high content of fluoride, which is statistically unlikely for ¹⁸F. However, it is worth considering that the radiofluorinated nGO-PEG exhibits a high stability in aqueous solution.

### 3.2.3. Inclusion of a Radiolabelled Fragment during the Preparation of the Nanoparticle

Generation of the nanoparticle in the presence of fluoride, or of a previously fluorinated constituent, is another approach to introduce a radioactive fluoride through a direct interaction with the constituents of the nanoparticle. Relevant illustrations of such an approach are provided in this section.

A first example where the fluoride anion is directly engaged is related to the synthesis of up-converting nanoparticles labelled with ¹⁸F by co-precipitation under vigorous stirring of NaNO₃, Bi(NO₃)₃, Yb(NO₃)₃, or Tb(NO₃)₃ in the presence of NH₄F and [¹⁸F]-NaF for 1 min at room temperature in air [72]. The stability of the radiolabelled nanoparticles was evaluated in fetal bovine serum for up to 4 h showing by rTLC no release of free [¹⁸F]-fluoride.

A related strategy, albeit involving a covalently bonded fluorine atom, has been applied for the formation of other radiolabelled nano-objects, namely [¹⁸F]-labelled liposomes. These are based on the radiosynthesis of fluorinated lipids that are then mixed with amphiphilic compounds. As such, [¹⁸F]-fluorodipalmitin 17 ([¹⁸F]-FDP) has been synthesized from its corresponding tosylate 17 (Figure 17), from which the radiolabelled liposomes where obtained upon addition of DPPC, DSPE-PEG2000, and cholesterol, followed by evaporation and sonication for 10 min at 50 °C after resuspension in a PBS solution, and finally filtration on polycarbonate membranes [30].

<figure><img src="image_20.png" /><figcaption>**Figure 17.** Synthesis of [¹⁸F]-fluorodipalmitin 18 ([¹⁸F]-FDP).</figcaption></figure>

In a similar approach, [¹⁸F]-FDP 18 has also been engaged in the formation of liposomes featuring arginine-rich peptides functionalized with lipophilic tails [35]. With these constituents, liposome formation occurred upon addition of a suspension of the mixture above with [¹⁸F]-FDP 18, sonication at 60 °C for 1 min, filtration through polycarbonate membrane and purification on a sephadex column. Of course, other radiofluorinated lipophilic derivatives have been engaged in related approaches.

Cholesterol-based liposomes have been obtained by mixing 10-cholesteryloxy-1-[¹⁸F] fluorodecane 19 with DSPC, DSPE/PEG2000, and cholesterol in chloroform (Figure 18), followed by evaporation and stirring for 30 min at 60 °C in HEPES buffer at pH 7.4 [37].

<figure><img src="image_21.png" /><figcaption>**Figure 18.** Structure of 10-cholesteryloxy-1-[¹⁸F]-fluorodecane 19.</figcaption></figure>
derivatives [131]. With this substrate, the radiofluorination was shown to occur upon heating for 1 h in a TFA aqueous solution (pH 5.5) at 80 °C. The reaction was reported as highly efficient (RCY over 96%). FT-IR investigation of [¹⁹F]-fluorinated nGO indicates a high content of fluoride, which is statistically unlikely for ¹⁸F. However, it is worth considering that the radiofluorinated nGO-PEG exhibits a high stability in aqueous solution.

### 3.2.3. Inclusion of a Radiolabelled Fragment during the Preparation of the Nanoparticle

Generation of the nanoparticle in the presence of fluoride, or of a previously fluorinated constituent, is another approach to introduce a radioactive fluoride through a direct interaction with the constituents of the nanoparticle. Relevant illustrations of a such an approach are provided in this section.

A first example where the fluoride anion is directly engaged is related to the synthesis of up-converting nanoparticles labelled with ¹⁸F by co-precipitation under vigorous stirring of NaNO₃, Bi(NO₃)₃, Yb(NO₃)₃, or Tb(NO₃)₃ in the presence of NH₄F and [¹⁸F]-NaF for 1 min at room temperature in air [72]. The stability of the radiolabelled nanoparticles was evaluated in fetal bovine serum for up to 4 h showing by rTLC no release of free [¹⁸F]-fluoride.

A related strategy, albeit involving a covalently bonded fluorine atom, has been applied for the formation of other radiolabelled nano-objects, namely [¹⁸F]-labelled liposomes. These are based on the radiosynthesis of fluorinated lipids that are then mixed with amphiphilic compounds. As such, [¹⁸F]-fluorodipalmitin 17 ([¹⁸F]-FDP) has been synthesized from its corresponding tosylate 17 (Figure 17), from which the radiolabelled liposomes where obtained upon addition of DPPC, DSPE-PEG2000, and cholesterol, followed by evaporation and sonication for 10 min at 50 °C after resuspension in a PBS solution, and finally filtration on polycarbonate membranes [30].

<figure><img src="image_20.png" /><figcaption>**Figure 17.** Synthesis of [¹⁸F]-fluorodipalmitin 18 ([¹⁸F]-FDP).</figcaption></figure>

In a similar approach, [¹⁸F]-FDP 18 has also been engaged in the formation of liposomes featuring arginine-rich peptides functionalized with lipophilic tails [35]. With these constituents, liposome formation occurred upon addition of a suspension of the mixture above with [¹⁸F]-FDP 18, sonication at 60 °C for 1 min, filtration through polycarbonate membrane and purification on a sephadex column. Of course, other radiofluorinated lipophilic derivatives have been engaged in related approaches.

Cholesterol-based liposomes have been obtained by mixing 10-cholesteryloxy-1-[¹⁸F] fluorodecane 19 with DSPC, DSPE/PEG2000, and cholesterol in chloroform (Figure 18), followed by evaporation and stirring for 30 min at 60 °C in HEPES buffer at pH 7.4 [37].

<figure><img src="image_21.png" /><figcaption>**Figure 18.** Structure of 10-cholesteryloxy-1-[¹⁸F]-fluorodecane 19.</figcaption></figure>
The radiofluorinated probe has been accessed by a standard $S_N2$ reaction of the corresponding mesylate in DMSO for 10 min at 165 °C, and a two-step Sep-Pak ($C_{18}$ and Silica) with a 1% radiochemical yield.

Different approaches of radiolabelling have been followed to access a range of radiofluorinated liposomes constituted of PEG or hyperbranched polyglycerol (hbPG) polymers and cholesterol [18]. The first one involved the preparation of $[^{18}F]$-fluorocholest-5-ene 21 by nucleophilic displacement of the methylsulfonate derivative of cholesterol 20 (Figure 19). The second and the third were based on a CuAAC reaction of alkyne-functionalized PEG- and hbPG-substituted cholesterol with $[^{18}F]$-TEG-$N_3$ 23 leading, respectively, to 24 and 1. Liposomes were then formulated with dioleoyl-$sn$-glycero-3-phosphatidylcholine (DOPC), cholesterol, and the radiolabelled copolymer with a lipid to cholesterol ratio of 60:40 (Figure 20).

<figure><img src="image_22.png" /><figcaption>**Figure 19.** Synthesis of $[^{18}F]$-fluorocholest-5-ene 21.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Figure 20.** Synthesis and CuAAC reactions of $[^{18}F]$-TEG-$N_3$ 23.</figcaption></figure>
The radiofluorinated probe has been accessed by a standard $S_N2$ reaction of the corresponding mesylate in DMSO for 10 min at 165 °C, and a two-step Sep-Pak ($C_{18}$ and Silica) with a 1% radiochemical yield.

Different approaches of radiolabelling have been followed to access a range of radiofluorinated liposomes constituted of PEG or hyperbranched polyglycerol (hbPG) polymers and cholesterol [18]. The first one involved the preparation of $[^{18}F]$-fluorocholest-5-ene 21 by nucleophilic displacement of the methylsulfonate derivative of cholesterol 20 (Figure 19). The second and the third were based on a CuAAC reaction of alkyne-functionalized PEG- and hbPG-substituted cholesterol with $[^{18}F]$-TEG-$N_3$ 23 leading, respectively, to 24 and 1. Liposomes were then formulated with dioleoyl-$sn$-glycero-3-phosphatidylcholine (DOPC), cholesterol, and the radiolabelled copolymer with a lipid to cholesterol ratio of 60:40 (Figure 20).

<figure><img src="image_22.png" /><figcaption>**Figure 19.** Synthesis of $[^{18}F]$-fluorocholest-5-ene 21.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Figure 20.** Synthesis and CuAAC reactions of $[^{18}F]$-TEG-$N_3$ 23.</figcaption></figure>