Similar radiolabelling has been performed using [¹⁸F]-TEG-N₃ 23 on linear and hyperbranched hexadecylbased polymers. The stability of the liposome was assessed, right after decay and after one month, by measuring the hydrodynamic radius of the liposomes. This was achieved at temperatures ranging from 20 to 50 °C by 5 °C increments, showing no evolution of the measured radii [23].

The use of general purpose radiolabelling prosthetic groups has also been investigated. As such, [¹⁸F]-Fluorobenzyl-2-bromoacetamide 27 ([¹⁸F]-FBBA) has been engaged in the radiolabelling of Paclitaxel-loaded polyester nanoparticles [45]. FBBA was obtained by fluorination of trimethylammoniumbenzonitrile 25 through a S<sub>N</sub>Ar displacement followed by LAH reduction of 26 and acylation with bromoacetyl bromide (Figure 21).

<figure><img src="image_24.png" /><figcaption>**Figure 21.** Synthesis of [¹⁸F]-FBBA 27.</figcaption></figure>

Formation of the polymer-based nanoparticle was achieved through the following three distinct strategies using different block-copolymers (Figure 22), which were shown to impact the distribution, metabolism, and elimination:

<figure><img src="image_25.png" /><figcaption>**Figure 22.** Block-copolymers involved in the formation of Paclitaxel-loaded and [¹⁸F]-FBBA-labelled nanoparticles.</figcaption></figure>

(i) using a mixture of radiolabelled block-copolymer 28 and one copolymer linked to Paclitaxel; (ii) using a mixture of block-copolymer 28, radiolabelled block-copolymer 29, and one copolymer linked to Paclitaxel; and (iii) a mixture of copolymer 28, FBBA, and one copolymer linked to Paclitaxel. Somehow expectedly, the radioactivity was progressively released from the third nano-object, most probably through water diffusion in the polymer core matrix.

A less-common approach towards radiolabelled gold nanoparticles has been performed through a double derivatization of [¹⁸F]-FDG 34 (Figure 23), pre- and post-radiolabelling [12].

<figure><img src="image_26.png" /><figcaption>**Figure 23.** Molecular structure of [¹⁸F]-FDG 30.</figcaption></figure>
Similar radiolabelling has been performed using [¹⁸F]-TEG-N₃ 23 on linear and hyperbranched hexadecylbased polymers. The stability of the liposome was assessed, right after decay and after one month, by measuring the hydrodynamic radius of the liposomes. This was achieved at temperatures ranging from 20 to 50 °C by 5 °C increments, showing no evolution of the measured radii [23].

The use of general purpose radiolabelling prosthetic groups has also been investigated. As such, [¹⁸F]-Fluorobenzyl-2-bromoacetamide 27 ([¹⁸F]-FBBA) has been engaged in the radiolabelling of Paclitaxel-loaded polyester nanoparticles [45]. FBBA was obtained by fluorination of trimethylammoniumbenzonitrile 25 through a S<sub>N</sub>Ar displacement followed by LAH reduction of 26 and acylation with bromoacetyl bromide (Figure 21).

<figure><img src="image_24.png" /><figcaption>**Figure 21.** Synthesis of [¹⁸F]-FBBA 27.</figcaption></figure>

Formation of the polymer-based nanoparticle was achieved through the following three distinct strategies using different block-copolymers (Figure 22), which were shown to impact the distribution, metabolism, and elimination:

<figure><img src="image_25.png" /><figcaption>**Figure 22.** Block-copolymers involved in the formation of Paclitaxel-loaded and [¹⁸F]-FBBA-labelled nanoparticles.</figcaption></figure>

(i) using a mixture of radiolabelled block-copolymer 28 and one copolymer linked to Paclitaxel; (ii) using a mixture of block-copolymer 28, radiolabelled block-copolymer 29, and one copolymer linked to Paclitaxel; and (iii) a mixture of copolymer 28, FBBA, and one copolymer linked to Paclitaxel. Somehow expectedly, the radioactivity was progressively released from the third nano-object, most probably through water diffusion in the polymer core matrix.

A less-common approach towards radiolabelled gold nanoparticles has been performed through a double derivatization of [¹⁸F]-FDG 34 (Figure 23), pre- and post-radiolabelling [12].

<figure><img src="image_26.png" /><figcaption>**Figure 23.** Molecular structure of [¹⁸F]-FDG 30.</figcaption></figure>
As illustrated in Figure 24, the process is initiated by the reaction of cysteamine with the peracetylated mannose triflate 31, precursor of the [¹⁸F]-FDG synthesis, followed by radiofluorination of 32 in DMF to provide 33. The gold nanoparticles 34 are generated from HAuCL₄ in the presence of NaBH₄ and 33, which is thus bound to gold through its thiol terminus. The acetates are cleaved under these reductive conditions. Upon reaction with carbonyldiimidazole (CDI), the released alcohols offer handles to conjugate antibodies such as in 35 for which anti-MTDH are favouring a vectorization to MCF7 breast cancer cells. Although this use of the [¹⁸F]-FDG scaffold to link the nanoparticle to an antibody appears highly original, the overall time of synthesis (almost 5 h) questions the global efficiency of the process from a radiochemical perspective.

<figure><img src="image_27.png" /><figcaption>**Figure 24.** [¹⁸F]-FDG-based generation of radiolabelled and vectorized gold nanoparticles 35.</figcaption></figure>

A simplified but similar strategy has been applied to the synthesis of magnetic iron oxide nanoparticles [148]. Indeed, addition of iron (III) chloride and sodium borohydride, at pH 9 and 60 °C, to a solution of the peracetylated [¹⁸F]-FDG-cysteamine derivative 37 allowed the isolation, after 2 h, of black magnetite (Fe₃O₄) nanoparticles, which could be separated using a magnet.

[¹⁸F]-succinimidylfluorobenzoate ([¹⁸F]-SFB) 38, another very common fluoroaromatic prosthetic group, has been used to radiolabel a wide range of nanoparticles. [¹⁸F]-SFB 38 is generally synthesized from the ammonium 35 after saponification of 36 and further activation using TSTU 37 as illustrated in Figure 25 [149].

Although [¹⁸F]-SFB 38 has been extensively used for couplings at the surface of preformed nanoparticles (see Section 3.3.2), it was also involved in one example of in vivo formation of radiolabelled nanoparticles. First, 38 was used to radiolabel the amino group of the lysine of an Ac-RVRRC(StBu)K peptide designed for detection of tumour cells exhibiting a furin overexpression. This lysine also featured a 2-cyanobenzothiazole moiety [19]. After injection, in vivo reduction in the disulfide (by GSH inside the cell) was followed by furin cleavage of the cell-penetrating peptide RVRR. This promoted the
As illustrated in Figure 24, the process is initiated by the reaction of cysteamine with the peracetylated mannose triflate 31, precursor of the [¹⁸F]-FDG synthesis, followed by radiofluorination of 32 in DMF to provide 33. The gold nanoparticles 34 are generated from HAuCL₄ in the presence of NaBH₄ and 33, which is thus bound to gold through its thiol terminus. The acetates are cleaved under these reductive conditions. Upon reaction with carbonyldiimidazole (CDI), the released alcohols offer handles to conjugate antibodies such as in 35 for which anti-MTDH are favouring a vectorization to MCF7 breast cancer cells. Although this use of the [¹⁸F]-FDG scaffold to link the nanoparticle to an antibody appears highly original, the overall time of synthesis (almost 5 h) questions the global efficiency of the process from a radiochemical perspective.

<figure><img src="image_27.png" /><figcaption>**Figure 24.** [¹⁸F]-FDG-based generation of radiolabelled and vectorized gold nanoparticles 35.</figcaption></figure>

A simplified but similar strategy has been applied to the synthesis of magnetic iron oxide nanoparticles [148]. Indeed, addition of iron (III) chloride and sodium borohydride, at pH 9 and 60 °C, to a solution of the peracetylated [¹⁸F]-FDG-cysteamine derivative 37 allowed the isolation, after 2 h, of black magnetite (Fe₃O₄) nanoparticles, which could be separated using a magnet.

[¹⁸F]-succinimidylfluorobenzoate ([¹⁸F]-SFB) 38, another very common fluoroaromatic prosthetic group, has been used to radiolabel a wide range of nanoparticles. [¹⁸F]-SFB 38 is generally synthesized from the ammonium 35 after saponification of 36 and further activation using TSTU 37 as illustrated in Figure 25 [149].

Although [¹⁸F]-SFB 38 has been extensively used for couplings at the surface of preformed nanoparticles (see Section 3.3.2), it was also involved in one example of in vivo formation of radiolabelled nanoparticles. First, 38 was used to radiolabel the amino group of the lysine of an Ac-RVRRC(StBu)K peptide designed for detection of tumour cells exhibiting a furin overexpression. This lysine also featured a 2-cyanobenzothiazole moiety [19]. After injection, in vivo reduction in the disulfide (by GSH inside the cell) was followed by furin cleavage of the cell-penetrating peptide RVRR. This promoted the
formation of a dimer via the condensation of the aminothiol of the cysteine terminus with the cyanobenzothiazole moiety of the lysine. Co-injection of the [¹⁹F]-analog favoured the formation of the radiolabelled dimers. These dimers self-assemble into nanoparticles inside the cells and, as they lack the cell membrane translocating ability of the RVRR fragment, they concentrate the radioactivity inside the tumour cell. Extension of this work has been carried out using the kit-like radiofluorination developed by the Perrin group [150], allowing a simple radiofluorination process based on an isotope exchange on a zwitterionic trifluoroborate (so called AMBF₃) [151].

<figure><img src="image_28.png" /><figcaption>**Figure 25.** Synthesis of [¹⁸F]-SFB 38.</figcaption></figure>

### 3.3. Introduction of Fluoride by Modification of the Surface of the Nanoparticle

These various methods consist in performing the nanoparticles, eventually preparing their surface for the radiolabelling, and then introducing the radioactive fluorine via an organic fragment. The differences can arise from the functionalization of the outer layer of the nanoparticles and from its ability to interact with the radiolabelled probe either through supramolecular-type interactions (including solid-phase transition methods for liposomes), as it will be first exemplified, or through covalent binding. In the latter case, we will first illustrate the common approach that relies on the radiofluorination of the nanoparticle prior to its injection, then we will discuss a more recent process based on bioorthogonal reactions that permits the in vivo labelling of the nanoparticles after their injection, biodistribution, and accumulation in the region of interest.

#### 3.3.1. Direct Introduction of a Radiofluorinated Adduct on the Nanoparticle Supramolecular Interaction

Some specific nanoparticles allow the integration of one radiolabelled constituent after their first generation. This is especially the case of liposomes and of some polymer-based nanoparticles. This differs practically from the “one-pot” generation and radiolabelling of similar nanoparticles described in the previous section by the possibility to isolate and fully characterize the nanoparticles before introduction of the radiofluorinated block. Yet for this post-synthetic approach, one can question the effect of the introduction of another constituent on the nature and properties of the nanoparticles, which might only be assessed after radioactive decay, limiting the exact characterization of the object used for in vivo exploration.

The radiolabelling of liposomes by solid-phase transition methods has been established as an efficient approach to introduce radiolabelled probes onto pre-formed liposomes [29]. The proof of concept was first established by a thorough identification of amphiphilic compounds (lipids + PEG) of high incorporation efficiency. The obtained liposomes were then incubated at 65 °C for 15 min with few [¹⁸F]-fluorinated amphiphilic compounds. This process, including repeated vortex mixing, allowed the generation of radiofluorinated liposomes with efficiencies ranging from 60 to 90%. Stability in foetal bovine serum proved highly dependent on the nature of the radiolabelled adduct with **39** (Figure 26) being the
formation of a dimer via the condensation of the aminothiol of the cysteine terminus with the cyanobenzothiazole moiety of the lysine. Co-injection of the [¹⁹F]-analog favoured the formation of the radiolabelled dimers. These dimers self-assemble into nanoparticles inside the cells and, as they lack the cell membrane translocating ability of the RVRR fragment, they concentrate the radioactivity inside the tumour cell. Extension of this work has been carried out using the kit-like radiofluorination developed by the Perrin group [150], allowing a simple radiofluorination process based on an isotope exchange on a zwitterionic trifluoroborate (so called AMBF₃) [151].

<figure><img src="image_28.png" /><figcaption>**Figure 25.** Synthesis of [¹⁸F]-SFB 38.</figcaption></figure>

### 3.3. Introduction of Fluoride by Modification of the Surface of the Nanoparticle

These various methods consist in performing the nanoparticles, eventually preparing their surface for the radiolabelling, and then introducing the radioactive fluorine via an organic fragment. The differences can arise from the functionalization of the outer layer of the nanoparticles and from its ability to interact with the radiolabelled probe either through supramolecular-type interactions (including solid-phase transition methods for liposomes), as it will be first exemplified, or through covalent binding. In the latter case, we will first illustrate the common approach that relies on the radiofluorination of the nanoparticle prior to its injection, then we will discuss a more recent process based on bioorthogonal reactions that permits the in vivo labelling of the nanoparticles after their injection, biodistribution, and accumulation in the region of interest.

#### 3.3.1. Direct Introduction of a Radiofluorinated Adduct on the Nanoparticle Supramolecular Interaction

Some specific nanoparticles allow the integration of one radiolabelled constituent after their first generation. This is especially the case of liposomes and of some polymer-based nanoparticles. This differs practically from the “one-pot” generation and radiolabelling of similar nanoparticles described in the previous section by the possibility to isolate and fully characterize the nanoparticles before introduction of the radiofluorinated block. Yet for this post-synthetic approach, one can question the effect of the introduction of another constituent on the nature and properties of the nanoparticles, which might only be assessed after radioactive decay, limiting the exact characterization of the object used for in vivo exploration.

The radiolabelling of liposomes by solid-phase transition methods has been established as an efficient approach to introduce radiolabelled probes onto pre-formed liposomes [29]. The proof of concept was first established by a thorough identification of amphiphilic compounds (lipids + PEG) of high incorporation efficiency. The obtained liposomes were then incubated at 65 °C for 15 min with few [¹⁸F]-fluorinated amphiphilic compounds. This process, including repeated vortex mixing, allowed the generation of radiofluorinated liposomes with efficiencies ranging from 60 to 90%. Stability in foetal bovine serum proved highly dependent on the nature of the radiolabelled adduct with **39** (Figure 26) being the
most appropriate, both in terms of incorporation efficiency and stability, and far superior
to the analogues featuring aromatic groups.

<figure><img src="image_29.png" /><figcaption>**Figure 26.** Amphiphilic derivative **39** for solid-phase transition radiofluorination of liposomes.</figcaption></figure>

Amphiphilic derivative **39** is obtained by a standard S<sub>N</sub>2-type procedure from its
corresponding tosylate. This approach has been applied in, e.g., liposome-encapsulating
hemoglobin (LEH) [31]. These have been labelled by incubation of LEH with 1-[<sup>18</sup>F]fluoro-
3,6-dioxatetracosane **40** with a labelling efficiency of 46 ± 5% (indicating that half of
the radioactivity remained in the supernatant upon incubation). However, the efficiency
of the radiolabelling of 3,6-dioxatetracosane tosylate (i.e., prior to the association of the
radiofluorinated probe **39** to LEH) should still be considered to evaluate the overall radiola-
belling efficiency.

The same authors have reported a similar approach to radiolabel another kind of lipo-
some made of disterearoylphosphatidylcholine, cholesterol, and distearoylphosphatidyle-
tanolaminePEG, with a PEG of 2000 g/mol with no further details than previously re-
ported [33].

Alternatively, the use of the biotin–avidin naturally occurring supramolecular bioconjugating process has also been investigated in order to perform the radiofluorination of PLGA nanoparticles [44]. To achieve this coupling, PLGA nanoparticles were functionalized with avidin. On the other side, a biotin radioconjugate **41** was synthesized using a fluorobenzylamine **40** obtained by BH<sub>3</sub>•SMe<sub>2</sub> reduction of the corresponding 4-fluorobenzonitrile (Figure 27). The radiofluorination of the latter was previously described for [<sup>18</sup>F]-FBBA **27** (Figure 21) to occur by S<sub>N</sub>Ar displacement of the trimethylammoniumbenzonitrile **25**.

<figure><img src="image_30.png" /><figcaption>**Figure 27.** Synthesis of the [<sup>18</sup>F]-radiolabelled biotin derivative **41**.</figcaption></figure>

Confirmation of the stability of the radiolabelling was performed by sonication of
the radiolabelled nanoparticles for 30 min and subsequent centrifugation: no remaining
radioactivity was detected in the supernatant. Yet one could question the solubility of the
fluorinated adduct NPB4 in the aqueous buffer, which could be trapped in the polymer
nanoparticle, or on its surface, but could be transferred to lipophilic biomolecules in vivo,
thus providing an off-target image.

Covalent Labelling via SN2-Type Reaction

In a related fashion, micelles made of a block-copolymer featuring mesylate leaving
groups on pendant alkyl chains have been submitted to a direct radiofluorination. This
has been achieved with [<sup>18</sup>F]-fluoride under standard S<sub>N</sub>2-type reaction of mesylates
most appropriate, both in terms of incorporation efficiency and stability, and far superior
to the analogues featuring aromatic groups.

<figure><img src="image_29.png" /><figcaption>**Figure 26.** Amphiphilic derivative **39** for solid-phase transition radiofluorination of liposomes.</figcaption></figure>

Amphiphilic derivative **39** is obtained by a standard S<sub>N</sub>2-type procedure from its
corresponding tosylate. This approach has been applied in, e.g., liposome-encapsulating
hemoglobin (LEH) [31]. These have been labelled by incubation of LEH with 1-[<sup>18</sup>F]fluoro-
3,6-dioxatetracosane **40** with a labelling efficiency of 46 ± 5% (indicating that half of
the radioactivity remained in the supernatant upon incubation). However, the efficiency
of the radiolabelling of 3,6-dioxatetracosane tosylate (i.e., prior to the association of the
radiofluorinated probe **39** to LEH) should still be considered to evaluate the overall radiola-
belling efficiency.

The same authors have reported a similar approach to radiolabel another kind of lipo-
some made of disterearoylphosphatidylcholine, cholesterol, and distearoylphosphatidyle-
tanolaminePEG, with a PEG of 2000 g/mol with no further details than previously re-
ported [33].

Alternatively, the use of the biotin–avidin naturally occurring supramolecular bioconjugating process has also been investigated in order to perform the radiofluorination of PLGA nanoparticles [44]. To achieve this coupling, PLGA nanoparticles were functionalized with avidin. On the other side, a biotin radioconjugate **41** was synthesized using a fluorobenzylamine **40** obtained by BH<sub>3</sub>•SMe<sub>2</sub> reduction of the corresponding 4-fluorobenzonitrile (Figure 27). The radiofluorination of the latter was previously described for [<sup>18</sup>F]-FBBA **27** (Figure 21) to occur by S<sub>N</sub>Ar displacement of the trimethylammoniumbenzonitrile **25**.

<figure><img src="image_30.png" /><figcaption>**Figure 27.** Synthesis of the [<sup>18</sup>F]-radiolabelled biotin derivative **41**.</figcaption></figure>

Confirmation of the stability of the radiolabelling was performed by sonication of
the radiolabelled nanoparticles for 30 min and subsequent centrifugation: no remaining
radioactivity was detected in the supernatant. Yet one could question the solubility of the
fluorinated adduct NPB4 in the aqueous buffer, which could be trapped in the polymer
nanoparticle, or on its surface, but could be transferred to lipophilic biomolecules in vivo,
thus providing an off-target image.

Covalent Labelling via SN2-Type Reaction

In a related fashion, micelles made of a block-copolymer featuring mesylate leaving
groups on pendant alkyl chains have been submitted to a direct radiofluorination. This
has been achieved with [<sup>18</sup>F]-fluoride under standard S<sub>N</sub>2-type reaction of mesylates
introduced at termini of PEG chains in the presence of BHT with a radiochemical conversion of 61% [50]. These radiofluorinated polymers, obtained from ROMP polymerization of norbornene-imides and cross-linked with cinnamoyl moieties, were shown to undergo a full displacement of the mesylate termini by further addition of [¹⁹F]-fluoride to avoid undesired reactivity of the mesylates in vivo.

### 3.3.2. Coupling of a Radiofluorinated Prosthetic Group at the Surface of a Nanoparticle

An alternative approach to perform the radiofluorination of nanoparticles consists in incorporating the prosthetic group to a pendant chemical function displayed at the surface of the nanoparticle.

*Ex Vivo Radiolabelling*

#### C-F-Based Derived Prosthetic Groups

A short [¹⁸F]-fluoroethyl-diethyleneglycol-ethylazide **43** has been involved in the CuAAC coupling with the alkyne termini of zinc oxide nanoparticles (Figure 28) [75,88]. The radiofluorinated adduct was classically obtained from **42** by displacement of a mesylate in acetonitrile at 100 °C in the presence of 37 MBq (1 mCi) of [¹⁸F]-tetrabutylammonium fluoride. It was purified by a short path silica gel chromatography. The stability of the obtained nanoparticles has been established by incubation both in saline and HCl solution, which led to no radioactivity release for up to 7 h, as identified by radio-TLC.

<figure><img src="image_31.png" /><figcaption>**Figure 28.** Synthesis of [¹⁸F]-fluoroethyl-diethyleneglycol-ethylazide **43**.</figcaption></figure>

The related [¹⁸F]-fluoroethyl-triethyleneglycol-ethylazide **44** (Figure 29) has been engaged after common radiofluorination in the CuAAC (in the presence of copper sulfate and sodium ascorbate) to label dextran-coated iron oxide nanoparticles featuring alkyne termini [76].

<figure><img src="image_32.png" /><figcaption>**Figure 29.** Elongated [¹⁸F]-fluoroethyl-PEG-azide **44**.</figcaption></figure>

This same approach had been previously explored with success for the generation of dextran-coated iron oxide nanoparticles [74].

A shorter prosthetic group such as [¹⁸F]-fluoroethyltosylate **46** has also been engaged in the radiolabelling of low and high molecular weight methacrylate-based polymers featuring derivatizations with folic acid and tyramine residues [40]. [¹⁸F]-Fluoroethyltosylate **46**, generated under conventional radiofluorination methods from **45** (Figure 30), was reacted with the phenol moiety of tyramine residues in the presence of sodium hydroxide, providing a 5 and 10% radiochemical yield for high and low molecular weight polymers, respectively.
introduced at termini of PEG chains in the presence of BHT with a radiochemical conversion of 61% [50]. These radiofluorinated polymers, obtained from ROMP polymerization of norbornene-imides and cross-linked with cinnamoyl moieties, were shown to undergo a full displacement of the mesylate termini by further addition of [¹⁹F]-fluoride to avoid undesired reactivity of the mesylates in vivo.

### 3.3.2. Coupling of a Radiofluorinated Prosthetic Group at the Surface of a Nanoparticle

An alternative approach to perform the radiofluorination of nanoparticles consists in incorporating the prosthetic group to a pendant chemical function displayed at the surface of the nanoparticle.

*Ex Vivo Radiolabelling*

#### C-F-Based Derived Prosthetic Groups

A short [¹⁸F]-fluoroethyl-diethyleneglycol-ethylazide **43** has been involved in the CuAAC coupling with the alkyne termini of zinc oxide nanoparticles (Figure 28) [75,88]. The radiofluorinated adduct was classically obtained from **42** by displacement of a mesylate in acetonitrile at 100 °C in the presence of 37 MBq (1 mCi) of [¹⁸F]-tetrabutylammonium fluoride. It was purified by a short path silica gel chromatography. The stability of the obtained nanoparticles has been established by incubation both in saline and HCl solution, which led to no radioactivity release for up to 7 h, as identified by radio-TLC.

<figure><img src="image_31.png" /><figcaption>**Figure 28.** Synthesis of [¹⁸F]-fluoroethyl-diethyleneglycol-ethylazide **43**.</figcaption></figure>

The related [¹⁸F]-fluoroethyl-triethyleneglycol-ethylazide **44** (Figure 29) has been engaged after common radiofluorination in the CuAAC (in the presence of copper sulfate and sodium ascorbate) to label dextran-coated iron oxide nanoparticles featuring alkyne termini [76].

<figure><img src="image_32.png" /><figcaption>**Figure 29.** Elongated [¹⁸F]-fluoroethyl-PEG-azide **44**.</figcaption></figure>

This same approach had been previously explored with success for the generation of dextran-coated iron oxide nanoparticles [74].

A shorter prosthetic group such as [¹⁸F]-fluoroethyltosylate **46** has also been engaged in the radiolabelling of low and high molecular weight methacrylate-based polymers featuring derivatizations with folic acid and tyramine residues [40]. [¹⁸F]-Fluoroethyltosylate **46**, generated under conventional radiofluorination methods from **45** (Figure 30), was reacted with the phenol moiety of tyramine residues in the presence of sodium hydroxide, providing a 5 and 10% radiochemical yield for high and low molecular weight polymers, respectively.
<figure><img src="image_33.png" /><figcaption>**Figure 30.** Synthesis of [¹⁸F]-fluoroethyltosylate 46.</figcaption></figure>

This [¹⁸F]-fluoroethyltosylate 46 was also engaged in the radiofluorination of polymeric nanoparticles. In this scenario, block-copolymers featuring one phenol fragment are alkylated by **46**. One early example of radiofluorination of a biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA) polymer has been achieved by modification of the polymer with tyramine [41]. This also allowed the radiofluorination by alkylation of the phenol moiety of the tyramine with **46**, obtained by S<sub>N</sub>2-type reaction between [¹⁸F]-fluoride and ethylene ditosylate **45** (Figure 30). The obtained polymer particles showed excellent stabilities, minimal metabolism, and an exclusive renal clearance evidenced by their accumulation in kidneys and the bladder. Extension of this work to HPMA polymer particles doped with lauryl methacrylate (random or as block-copolymer) demonstrated a linear variation in tumour uptake and molecular weight of the polymers. Thus, it illustrated the need to individualize polymer-based radiotherapy as tumour uptake is strongly related with the tumour model [42].

Another short prosthetic group, namely [¹⁸F]-fluoroethylazide **48**, has been obtained from its corresponding tosylate **47**, using the standard S<sub>N</sub>2-type radiofluorination method (Figure 31) [78].

<figure><img src="image_34.png" /><figcaption>**Figure 31.** Synthesis of [¹⁸F]-fluoroethylazide **48**.</figcaption></figure>

This radiolabelled fragment was then engaged in a CuAAC reaction (in the presence of copper sulfate and sodium ascorbate) with the alkyne termini of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coated with a polyacrylic polymer, then with polydopamine, and also featuring a cyclic RGDfK terminus for vectorization. The key alkyne functionalization of the USPIO was achieved by incubation with an amino-PEG-alkyne, in which the amine reacts via a Michael reaction with the quinone moiety of the polydopamine coating.

Reversing the partners of the CuAAC reaction, organic nanoparticles composed of 40% polyglucose and 60% L-lysine functionalized with azide termini have been radiolabelled with a propargylated [¹⁸F]-fluoroethoxydiethylene glycol 50 [152]. The latter was classically prepared from its corresponding tosylate **49** and purified by preparative HPLC to provide 56% RCY of the radiolabelled alkyne intermediate (Figure 32), which after CuAAC coupling provided the organic nanoparticles in 38% decay-corrected RCY in almost 2 h. When performed from 37 GBq of [¹⁸F]-fluoride in a hot cell, the method gave access to the radiofluorinated nanoparticles in lower yield (13%), probably related to the lower yield achieved for the alkyne intermediate, which was then carried out in DMSO in 23% RCY.
<figure><img src="image_33.png" /><figcaption>**Figure 30.** Synthesis of [¹⁸F]-fluoroethyltosylate 46.</figcaption></figure>

This [¹⁸F]-fluoroethyltosylate 46 was also engaged in the radiofluorination of polymeric nanoparticles. In this scenario, block-copolymers featuring one phenol fragment are alkylated by **46**. One early example of radiofluorination of a biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA) polymer has been achieved by modification of the polymer with tyramine [41]. This also allowed the radiofluorination by alkylation of the phenol moiety of the tyramine with **46**, obtained by S<sub>N</sub>2-type reaction between [¹⁸F]-fluoride and ethylene ditosylate **45** (Figure 30). The obtained polymer particles showed excellent stabilities, minimal metabolism, and an exclusive renal clearance evidenced by their accumulation in kidneys and the bladder. Extension of this work to HPMA polymer particles doped with lauryl methacrylate (random or as block-copolymer) demonstrated a linear variation in tumour uptake and molecular weight of the polymers. Thus, it illustrated the need to individualize polymer-based radiotherapy as tumour uptake is strongly related with the tumour model [42].

Another short prosthetic group, namely [¹⁸F]-fluoroethylazide **48**, has been obtained from its corresponding tosylate **47**, using the standard S<sub>N</sub>2-type radiofluorination method (Figure 31) [78].

<figure><img src="image_34.png" /><figcaption>**Figure 31.** Synthesis of [¹⁸F]-fluoroethylazide **48**.</figcaption></figure>

This radiolabelled fragment was then engaged in a CuAAC reaction (in the presence of copper sulfate and sodium ascorbate) with the alkyne termini of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coated with a polyacrylic polymer, then with polydopamine, and also featuring a cyclic RGDfK terminus for vectorization. The key alkyne functionalization of the USPIO was achieved by incubation with an amino-PEG-alkyne, in which the amine reacts via a Michael reaction with the quinone moiety of the polydopamine coating.

Reversing the partners of the CuAAC reaction, organic nanoparticles composed of 40% polyglucose and 60% L-lysine functionalized with azide termini have been radiolabelled with a propargylated [¹⁸F]-fluoroethoxydiethylene glycol 50 [152]. The latter was classically prepared from its corresponding tosylate **49** and purified by preparative HPLC to provide 56% RCY of the radiolabelled alkyne intermediate (Figure 32), which after CuAAC coupling provided the organic nanoparticles in 38% decay-corrected RCY in almost 2 h. When performed from 37 GBq of [¹⁸F]-fluoride in a hot cell, the method gave access to the radiofluorinated nanoparticles in lower yield (13%), probably related to the lower yield achieved for the alkyne intermediate, which was then carried out in DMSO in 23% RCY.
<figure><img src="image_35.png" /><figcaption>**Figure 32.** Synthesis of propargylated [¹⁸F]-fluoroethoxydiethylene glycol 50.</figcaption></figure>

A related method has been employed to perform the radiofluorination of iron oxide nanoparticles featuring surface functionalization with catechols bearing either a folic acid or an azido acetyl group [79]. In this case, two paths were investigated. One was based on a late radiolabelling strategy, with a coupling occurring between the azide and 5-tosyl-pentyne, followed by nucleophilic displacement of the tosyl by [¹⁸F]-fluoride under conventional conditions. The other, in a two-step process, required the preliminary formation of 5-[¹⁸F]-fluoropentyne from the corresponding 5-tosyl-pentyne, followed by the CuAAC to the azide carried by the nanoparticle catechol ligand. The former proved more efficient, which could be related to the known low boiling point of 5-[¹⁸F]-fluoropentyne [153].

In order to take advantage of the facile formation of amide bonds, NFP ([¹⁸F]-fluoropropionate *p*-nitrophenol), an activated [¹⁸F]-fluorinated ester, has been involved in the overall labelling of QDs [97]. For that purpose, amine-modified quantum dots have been submitted to multiplex functionalization through a tertiary linker 51, thus allowing the conjugation of bombesin (to target Gastrin-releasing peptide receptor) and RGD (to target integrin $\alpha_V\beta_3$) (Figure 33), as discussed in Section 2.2.3, and a further reaction with [¹⁸F]-NFP 54.

<figure><img src="image_36.png" /><figcaption>**Figure 33.** Structure of tertiary linker for vectorization and radiofluorination of QDs.</figcaption></figure>

[¹⁸F]-NFP 54 has been obtained through a three-step route starting with standard fluorination (Figure 34), followed by saponification and ester activation with bis-4-nitrophenylcarbonate at 100 °C for 10 min.

<figure><img src="image_37.png" /><figcaption>**Figure 34.** Synthesis of [¹⁸F]-NFP 54.</figcaption></figure>

Prosthetic groups featuring [¹⁸F]-(het)aryl fluorides are also frequently reported. Among the examples available, (2-fluoropyrid-3-yloxy)propylmaleimide, FPyME 59, in
<figure><img src="image_35.png" /><figcaption>**Figure 32.** Synthesis of propargylated [¹⁸F]-fluoroethoxydiethylene glycol 50.</figcaption></figure>

A related method has been employed to perform the radiofluorination of iron oxide nanoparticles featuring surface functionalization with catechols bearing either a folic acid or an azido acetyl group [79]. In this case, two paths were investigated. One was based on a late radiolabelling strategy, with a coupling occurring between the azide and 5-tosyl-pentyne, followed by nucleophilic displacement of the tosyl by [¹⁸F]-fluoride under conventional conditions. The other, in a two-step process, required the preliminary formation of 5-[¹⁸F]-fluoropentyne from the corresponding 5-tosyl-pentyne, followed by the CuAAC to the azide carried by the nanoparticle catechol ligand. The former proved more efficient, which could be related to the known low boiling point of 5-[¹⁸F]-fluoropentyne [153].

In order to take advantage of the facile formation of amide bonds, NFP ([¹⁸F]-fluoropropionate *p*-nitrophenol), an activated [¹⁸F]-fluorinated ester, has been involved in the overall labelling of QDs [97]. For that purpose, amine-modified quantum dots have been submitted to multiplex functionalization through a tertiary linker 51, thus allowing the conjugation of bombesin (to target Gastrin-releasing peptide receptor) and RGD (to target integrin $\alpha_V\beta_3$) (Figure 33), as discussed in Section 2.2.3, and a further reaction with [¹⁸F]-NFP 54.

<figure><img src="image_36.png" /><figcaption>**Figure 33.** Structure of tertiary linker for vectorization and radiofluorination of QDs.</figcaption></figure>

[¹⁸F]-NFP 54 has been obtained through a three-step route starting with standard fluorination (Figure 34), followed by saponification and ester activation with bis-4-nitrophenylcarbonate at 100 °C for 10 min.

<figure><img src="image_37.png" /><figcaption>**Figure 34.** Synthesis of [¹⁸F]-NFP 54.</figcaption></figure>

Prosthetic groups featuring [¹⁸F]-(het)aryl fluorides are also frequently reported. Among the examples available, (2-fluoropyrid-3-yloxy)propylmaleimide, FPyME 59, in
which three-step synthesis was previously described for protein radiofluorination (Figure 35) [154], has been engaged in the labelling of thiol termini containing QDs. These thiol termini were obtained by converting the amino groups present in the PEG phospholipid micelles, which encapsulated the QDs [16]. Fluorination was carried out on 56, followed by Boc deprotection yielding 57. Introduction of the imide moiety was performed by exchange with 58 providing, after purification by HPLC and evaporation of the solvents, $[^{18}F]$-FPyME 59.

<figure><img src="image_38.png" /><figcaption>**Figure 35.** Synthesis of $[^{18}F]$-FPyMe 58.</figcaption></figure>

Among the array of prosthetic groups, $[^{18}F]$-SFB 38 has been the most employed one. A first example reported the radiofluorination of nanodiamonds. These nanodiamonds were first treated by Fenton oxidation and surface-modified with aminopropyltriethoxysilane. Coupling of the surface amino groups with $[^{18}F]$-SFB 38 afforded the radiolabelled nanoparticles (Figure 36) [130].

<figure><img src="image_39.png" /><figcaption>**Figure 36.** $[^{18}F]$-radiolabelling of nanodiamonds.</figcaption></figure>

$[^{18}F]$-SFB 38 was also found to demonstrate successful application for the radiolabelling of gold nanoparticles functionalized with cysteine-lysine (CK) dipeptide and a CLPFFD peptide for brain penetration [120]. Once classically synthesized and purified by semipreparative HPLC, $[^{18}F]$-SFB 38 was dried and resuspended with a DMSO/sodium citrate solution of the gold nanoparticles, with stirring prolonged for 1 h at 50 °C. This yielded the radiolabelled nanoparticles with 0.8% RCY, thus exhibiting a limited efficiency. Their stability was assessed from incubation in plasma followed by dialysis and UV-Vis measurements showing a perfect stability over a 2 h period and a slight decrease over 1 d. This was somehow confirmed using the high sensitivity of the radioactivity detection combined with centrifugation and counting of the supernatant and the solid, which showed a release of 25% of the radioactivity in the supernatant.

$[^{18}F]$-SFB 38 has also been engaged for the radiolabelling of mesoporous silica nanoparticles functionalized with amino groups by way of sonication of colloidal silica nanoparticles with aminopropyltriethoxysilane [143]. The combination of the $[^{18}F]$-SFB 38 probe with the amino groups of these nanoparticles has been achieved by reaction of the two partners in a
which three-step synthesis was previously described for protein radiofluorination (Figure 35) [154], has been engaged in the labelling of thiol termini containing QDs. These thiol termini were obtained by converting the amino groups present in the PEG phospholipid micelles, which encapsulated the QDs [16]. Fluorination was carried out on 56, followed by Boc deprotection yielding 57. Introduction of the imide moiety was performed by exchange with 58 providing, after purification by HPLC and evaporation of the solvents, $[^{18}F]$-FPyME 59.

<figure><img src="image_38.png" /><figcaption>**Figure 35.** Synthesis of $[^{18}F]$-FPyMe 58.</figcaption></figure>

Among the array of prosthetic groups, $[^{18}F]$-SFB 38 has been the most employed one. A first example reported the radiofluorination of nanodiamonds. These nanodiamonds were first treated by Fenton oxidation and surface-modified with aminopropyltriethoxysilane. Coupling of the surface amino groups with $[^{18}F]$-SFB 38 afforded the radiolabelled nanoparticles (Figure 36) [130].

<figure><img src="image_39.png" /><figcaption>**Figure 36.** $[^{18}F]$-radiolabelling of nanodiamonds.</figcaption></figure>

$[^{18}F]$-SFB 38 was also found to demonstrate successful application for the radiolabelling of gold nanoparticles functionalized with cysteine-lysine (CK) dipeptide and a CLPFFD peptide for brain penetration [120]. Once classically synthesized and purified by semipreparative HPLC, $[^{18}F]$-SFB 38 was dried and resuspended with a DMSO/sodium citrate solution of the gold nanoparticles, with stirring prolonged for 1 h at 50 °C. This yielded the radiolabelled nanoparticles with 0.8% RCY, thus exhibiting a limited efficiency. Their stability was assessed from incubation in plasma followed by dialysis and UV-Vis measurements showing a perfect stability over a 2 h period and a slight decrease over 1 d. This was somehow confirmed using the high sensitivity of the radioactivity detection combined with centrifugation and counting of the supernatant and the solid, which showed a release of 25% of the radioactivity in the supernatant.

$[^{18}F]$-SFB 38 has also been engaged for the radiolabelling of mesoporous silica nanoparticles functionalized with amino groups by way of sonication of colloidal silica nanoparticles with aminopropyltriethoxysilane [143]. The combination of the $[^{18}F]$-SFB 38 probe with the amino groups of these nanoparticles has been achieved by reaction of the two partners in a
mixture of DMSO and sodium citrate buffer at pH 11 for 45 min, followed by a dilution with water and three iterative centrifugations providing an overall radiochemical yield of 14%. Similarly, [¹⁸F]-SFB **38** has also been used as a radiofluorinated probe to label polymeric nanoparticles made of acryloylated BSA coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-amino-propyl) methacrylamide hydrochloride (APM) [155]. The amino residue of the latter was used to both introduce an RGD-A7R peptide and [¹⁸F]-SFB **38**, at room temperature for 30 min in a borax buffer (pH 8.5). Incubation of the radiolabelled nanoparticles in PBS and calf serum exhibited a lower stability in the latter as identified by thin layer chromatography. The efficient reactivity of [¹⁸F]-SFB **38** with the amino termini of (aminopropyl)triethoxysilane-functionalized surfaces of nanoparticles has also been applied for the radiofluoration of ceria nanoparticles [89]. The reaction was shown to occur at room temperature within 1 h in a 3:2 mixture of DMSO and phosphate buffer (pH = 7.4). After dilution and centrifugation, the radiofluorinated nanoparticles were obtained with 18% RCY.

[¹⁸F]-SFB **38** has also been involved in the radiolabelling of a range of polymers featuring polyester grafted with chitosan, polylysine, or phosphatidylserine and calcium phosphate by incubation at 37 °C for 30 min [47]. Although the description of the formation of the polymer nanoparticles remains elusive, their labelling allowed a detailed in vivo investigation of their tumour-targeting ability.

4-[¹⁸F]-fluorobenzamido-N-ethylmaleimide ([¹⁸F]-FBEM **62**) previously described for labelling thiol-containing biomolecules (Figure 37) [156], has been engaged in the radiolabelling of sphingomyelin nanoemulsions functionalized with a thiol group [12]. The synthesis of [¹⁸F]-FBEM **62** was achieved from **35** through three steps via the intermediate **59**, which reacts with maleimide derivative **60** and the DEPC coupling agent **61**, in an automatized procedure. The synthesis required HPLC purification followed by solvent evaporation as previously performed for biomacromolecules, illustrating a direct parallel between the labelling of biomacromolecules and nano-objects.

<figure><img src="image_40.png" /><figcaption>**Figure 37.** Synthesis of [¹⁸F]-FBEM **62**.</figcaption></figure>

Yet the multistep character of this process limits its wide availability and might account for its scarce use for the radiofluorination of nanoparticles.

A more innovative approach based on the recent developments of conjugation involving the cycloaddition of strained multiple carbon-carbon bonds is nitrone functionalized gold nanoparticles, which have been radiofluorinated by way of strain-promoted alkyne-nitrone cycloaddition (SPANC) (Figure 38) [124]. The counterpart of the coupling reaction was a fluorinated cyclooctyne **67** obtained in a three-step post-radiofluorination fashion from **63** with an overall 22% RCY (decay corr.). After SPANC coupling, the radiofluorinated gold nanoparticles **68** were obtained with 6% RCY (decay corr.) after purification by size-exclusion chromatography.
mixture of DMSO and sodium citrate buffer at pH 11 for 45 min, followed by a dilution with water and three iterative centrifugations providing an overall radiochemical yield of 14%. Similarly, [¹⁸F]-SFB **38** has also been used as a radiofluorinated probe to label polymeric nanoparticles made of acryloylated BSA coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-amino-propyl) methacrylamide hydrochloride (APM) [155]. The amino residue of the latter was used to both introduce an RGD-A7R peptide and [¹⁸F]-SFB **38**, at room temperature for 30 min in a borax buffer (pH 8.5). Incubation of the radiolabelled nanoparticles in PBS and calf serum exhibited a lower stability in the latter as identified by thin layer chromatography. The efficient reactivity of [¹⁸F]-SFB **38** with the amino termini of (aminopropyl)triethoxysilane-functionalized surfaces of nanoparticles has also been applied for the radiofluoration of ceria nanoparticles [89]. The reaction was shown to occur at room temperature within 1 h in a 3:2 mixture of DMSO and phosphate buffer (pH = 7.4). After dilution and centrifugation, the radiofluorinated nanoparticles were obtained with 18% RCY.

[¹⁸F]-SFB **38** has also been involved in the radiolabelling of a range of polymers featuring polyester grafted with chitosan, polylysine, or phosphatidylserine and calcium phosphate by incubation at 37 °C for 30 min [47]. Although the description of the formation of the polymer nanoparticles remains elusive, their labelling allowed a detailed in vivo investigation of their tumour-targeting ability.

4-[¹⁸F]-fluorobenzamido-N-ethylmaleimide ([¹⁸F]-FBEM **62**) previously described for labelling thiol-containing biomolecules (Figure 37) [156], has been engaged in the radiolabelling of sphingomyelin nanoemulsions functionalized with a thiol group [12]. The synthesis of [¹⁸F]-FBEM **62** was achieved from **35** through three steps via the intermediate **59**, which reacts with maleimide derivative **60** and the DEPC coupling agent **61**, in an automatized procedure. The synthesis required HPLC purification followed by solvent evaporation as previously performed for biomacromolecules, illustrating a direct parallel between the labelling of biomacromolecules and nano-objects.

<figure><img src="image_40.png" /><figcaption>**Figure 37.** Synthesis of [¹⁸F]-FBEM **62**.</figcaption></figure>

Yet the multistep character of this process limits its wide availability and might account for its scarce use for the radiofluorination of nanoparticles.

A more innovative approach based on the recent developments of conjugation involving the cycloaddition of strained multiple carbon-carbon bonds is nitrone functionalized gold nanoparticles, which have been radiofluorinated by way of strain-promoted alkyne-nitrone cycloaddition (SPANC) (Figure 38) [124]. The counterpart of the coupling reaction was a fluorinated cyclooctyne **67** obtained in a three-step post-radiofluorination fashion from **63** with an overall 22% RCY (decay corr.). After SPANC coupling, the radiofluorinated gold nanoparticles **68** were obtained with 6% RCY (decay corr.) after purification by size-exclusion chromatography.
<figure><img src="image_41.png" /><figcaption>**Figure 38.** SPANC-based [¹⁸F]-radiolabelling of gold nanoparticles.</figcaption></figure>

Another, even more straightforward route, makes use of the [¹⁸F]-FDG scaffold. It takes advantage of the high reactivity of the aldehyde of the sugar with hydroxylamino groups to form oxime under aqueous conditions [77]. Thus, for the radiolabelling of iron oxide nanoparticles coated with dextran and functionalized with amino-oxyacetate termini, a simple incubation at 60 °C in PBS for 10 min with routinely prepared and clinically available [¹⁸F]-FDG allowed the formation of the radiolabelled nanoparticles in 24% radiochemical yield (decay corrected) in 40 min. Although stable for more than 1 h in water, these radiolabelled nanoparticles were found to be rather unstable in plasma as 50% of the radioactivity was released from the nanoparticles in 30 min, which might be due to dextran-coating fragmentation.

## Al-F-Derived Prosthetic Groups

Moving away from C–F bond-based prosthetic groups, chelated Al–F derivatives have also been employed for the ex vivo radiofluorination of nanoparticles. One relevant application concerns the radiolabelling of iron oxide nanoparticles coated with oleylamine-branched polyacrylic acid conjugated with a NOTA macrocycle [80]. A simple radiofluorination procedure was performed by incubation of the NOTA-conjugated nanoparticles in a sodium acetate buffer with a pre-heated solution of [¹⁸F]-NaF and AlCl₃ at 95 °C for 5 min. Purification was achieved by size-exclusion chromatography. The stability of the construct was assessed on ¹⁹F isotopologous nanoparticles by measuring via ICP-MS the amount of aluminium released upon incubation in PBS buffer. A high stability was determined as only 5% of the aluminium was released, and this occurred only during the first 2 h.

## Si-F-Based Derived Prosthetic Groups

The radiolabelling of silicon-based prosthetic groups, previously described for large biomolecule radiofluorination [157], has also been applied for the radiofluorination of gold nanoparticles (Figure 39) [122]. This has been achieved by addition of the thiophenol of SiFA derivative 70 to the maleimide termini on the gold nanoparticle 69 while cysteine-containing TATE was added to other maleimide on the surface to perform the vectorization of the nanoparticle 71. Fluorination of the SiFA group 70 occurred through a kit-like isotope exchange reaction.
<figure><img src="image_41.png" /><figcaption>Figure 38. SPANC-based [¹⁸F]-radiolabelling of gold nanoparticles.</figcaption></figure>

Another, even more straightforward route, makes use of the [¹⁸F]-FDG scaffold. It takes advantage of the high reactivity of the aldehyde of the sugar with hydroxylamino groups to form oxime under aqueous conditions [77]. Thus, for the radiolabelling of iron oxide nanoparticles coated with dextran and functionalized with amino-oxyacetate termini, a simple incubation at 60 °C in PBS for 10 min with routinely prepared and clinically available [¹⁸F]-FDG allowed the formation of the radiolabelled nanoparticles in 24% radiochemical yield (decay corrected) in 40 min. Although stable for more than 1 h in water, these radiolabelled nanoparticles were found to be rather unstable in plasma as 50% of the radioactivity was released from the nanoparticles in 30 min, which might be due to dextran-coating fragmentation.

## Al-F-Derived Prosthetic Groups

Moving away from C–F bond-based prosthetic groups, chelated Al–F derivatives have also been employed for the ex vivo radiofluorination of nanoparticles. One relevant application concerns the radiolabelling of iron oxide nanoparticles coated with oleylamine-branched polyacrylic acid conjugated with a NOTA macrocycle [80]. A simple radiofluorination procedure was performed by incubation of the NOTA-conjugated nanoparticles in a sodium acetate buffer with a pre-heated solution of [¹⁸F]-NaF and AlCl₃ at 95 °C for 5 min. Purification was achieved by size-exclusion chromatography. The stability of the construct was assessed on ¹⁹F isotopologous nanoparticles by measuring via ICP-MS the amount of aluminium released upon incubation in PBS buffer. A high stability was determined as only 5% of the aluminium was released, and this occurred only during the first 2 h.

## Si-F-Based Derived Prosthetic Groups

The radiolabelling of silicon-based prosthetic groups, previously described for large biomolecule radiofluorination [157], has also been applied for the radiofluorination of gold nanoparticles (Figure 39) [122]. This has been achieved by addition of the thiophenol of SiFA derivative 70 to the maleimide termini on the gold nanoparticle 69 while cysteine-containing TATE was added to other maleimide on the surface to perform the vectorization of the nanoparticle 71. Fluorination of the SiFA group 70 occurred through a kit-like isotope exchange reaction.
<figure><img src="image_42.png" /><figcaption>**Figure 39.** SiFA-based radiofluorination of gold nanoparticles.</figcaption></figure>

The same SiF- based strategy was more recently applied for the radiofluorination of polymeric core–shell nanoparticles [49]. Here again the isotope exchange method proved efficient as radiofluorinated nanoparticles were obtained in 53 to 72% RCY.

### P-F-Based Derived Prosthetic Groups

Following on isotope exchange-based radiofluorination of phosphorous compounds [158], a nicely innovative approach to radiofluorination of phosphorus-based prosthetic groups has been recently reported and adapted for the radiofluorination of PEGylated Pd nanosheets (Pd PEGs) [125]. The method is based on the nucleophilic ring opening of **72** by [¹⁸F]–fluoride under non-conventional aqueous conditions (MeCN/H₂O: 9/1) (Figure 40). This provides, after release of thiirane, the radiofluorinated phosphorofluoridodithioate **73**, in which sulfhydryl groups form stable coordination bonds with Pd-PEGs. However, stability investigations were not thoroughly described.

<figure><img src="image_43.png" /><figcaption>**Figure 40.** Synthesis of phosphorofluoridodithioate **73**.</figcaption></figure>

To conclude, coupling a radiofluorinated probe at the surface of a nanoparticle shares numerous common features with the labelling of biomacromolecules. Conventional C–F–based prosthetic groups, as more innovative approaches have been taking advantage of the fast an efficient labelling of main group elements, have been involved in the radiofluorination of nanoparticles. It clearly and obviously appears that the most straightforward methods to achieve the synthesis of radiolabelled prosthetic groups are the most appropriate, and that multistep approaches shall be difficult to implement in a systematic fashion. Once the radiolabelling technique is chosen, numerous coupling methods can be adapted
<figure><img src="image_42.png" /><figcaption>**Figure 39.** SiFA-based radiofluorination of gold nanoparticles.</figcaption></figure>

The same SiF-based strategy was more recently applied for the radiofluorination of polymeric core-shell nanoparticles [49]. Here again the isotope exchange method proved efficient as radiofluorinated nanoparticles were obtained in 53 to 72% RCY.

### P-F-Based Derived Prosthetic Groups

Following on isotope exchange-based radiofluorination of phosphorous compounds [158], a nicely innovative approach to radiofluorination of phosphorus-based prosthetic groups has been recently reported and adapted for the radiofluorination of PEGylated Pd nanosheets (Pd PEGs) [125]. The method is based on the nucleophilic ring opening of **72** by [¹⁸F]-fluoride under non-conventional aqueous conditions (MeCN/H₂O: 9/1) (Figure 40). This provides, after release of thiirane, the radiofluorinated phosphorofluoridodithioate **73**, in which sulfhydryl groups form stable coordination bonds with Pd-PEGs. However, stability investigations were not thoroughly described.

<figure><img src="image_43.png" /><figcaption>**Figure 40.** Synthesis of phosphorofluoridodithioate **73**.</figcaption></figure>

To conclude, coupling a radiofluorinated probe at the surface of a nanoparticle shares numerous common features with the labelling of biomacromolecules. Conventional C–F–based prosthetic groups, as more innovative approaches have been taking advantage of the fast an efficient labelling of main group elements, have been involved in the radiofluorination of nanoparticles. It clearly and obviously appears that the most straightforward methods to achieve the synthesis of radiolabelled prosthetic groups are the most appropriate, and that multistep approaches shall be difficult to implement in a systematic fashion. Once the radiolabelling technique is chosen, numerous coupling methods can be adapted
as soon as their kinetics and chemical efficiencies are high (to avoid long reaction times and achieve high radiochemical yields).

This parallel between biomacromolecules and nanoparticles is similarly observed by the development of highly innovative in vivo labelling techniques as illustrated below.

*In Vivo Radiolabelling*

A non-canonical radiolabelling method was engaged for in vivo radiofluorination of cancer cells pre-treated with GdF₃-HSA-tetrazine nanoparticles. In this case, addition of [¹⁸F]-FAI-NOTA complexes featuring a Reppe anhydride **74** (Figure 41) allowed the in vivo orthogonal bioconjugation [61].

<figure><img src="image_44.png" /><figcaption>**Figure 41.** [¹⁸F]-FAI-NOTA Reppe anhydride adduct for in vivo coupling.</figcaption></figure>

Another in vivo application of the strained promoted reactions, in this case a alkyne-azide cycloaddition, has been reported to occur between a radiofluorinated azide and azadibenzocyclooctyne functionalizing the surface of mesoporous silica nanoparticles [144]. Indeed, after the latter were injected and passively accumulated in tumours, they were shown to react with radiofluorinated azido-PEG **44** based on both PET imaging and necropsy data. The fluorination is achieved classically but in this case **44** is injected (2 h after the nanoparticles) and reacts in vivo.

A related approach has been applied for the in vivo radiofluorination of porous silicon nanoparticles [138] pre-targeted with a *trans*-cyclooctene (TCO) moiety. In vivo, the nanoparticle-carried TCO reacted with **78** made of a [¹⁸F]-5-fluoro-5-deoxy-ribose equipped with a tetrazine terminus. The synthesis of **78** is described in Figure 42. In a first step, [¹⁸F]-5-fluoro-5-deoxy-ribose **76** is obtained by a classical tosylate displacement from **75** in the presence of [¹⁸F]-fluoride, K₂CO₃, and K₂,2,2, followed by HPLC purification and acidic hydrolysis. In a second step, a tetrazine decorated with an aminoxy function **77** allowed a straightforward oxime linkage with **76** to form **78**. This oxime was then purified again by HPLC. Although the production of the tetrazine derivative appears somehow cumbersome, this approach represents an illustration of the pre-targeting concepts for in vivo labelling of nanoparticles.

To conclude on this overview of the methods involved for the radiolabelling of nanoparticles, as for most radiofluorinations, the smartest approach will consist in application of the "keep it simple" principle. Thus, late radiofluorinations, avoiding tedious purification of any [¹⁸F]-containing intermediates, shall be preferred. In this perspective, one can forecast an increasing use of the radiolabelling techniques based on main group elements such as alumina, silicon, and boron. The radiofluorination of metal-based nanoparticles featuring oxygen atoms might also take advantage of their high thermal stabilities. The generation of such nanoparticles incorporating [¹⁸O]-oxygen atoms can allow the formation of ¹⁸F through the nuclear ¹⁸O(p,n)¹⁸F by direct bombardment of the nanoparticles. Yet in this case the nature of the metal has to be taken into account to avoid the generation of long-lived isotopes through nuclear reaction of the metal itself.
as soon as their kinetics and chemical efficiencies are high (to avoid long reaction times and achieve high radiochemical yields).

This parallel between biomacromolecules and nanoparticles is similarly observed by the development of highly innovative in vivo labelling techniques as illustrated below.

*In Vivo Radiolabelling*

A non-canonical radiolabelling method was engaged for in vivo radiofluorination of cancer cells pre-treated with GdF₃-HSA-tetrazine nanoparticles. In this case, addition of [¹⁸F]-FAI-NOTA complexes featuring a Reppe anhydride **74** (Figure 41) allowed the in vivo orthogonal bioconjugation [61].

<figure><img src="image_44.png" /><figcaption>**Figure 41.** [¹⁸F]-FAI-NOTA Reppe anhydride adduct for in vivo coupling.</figcaption></figure>

Another in vivo application of the strained promoted reactions, in this case a alkyne-azide cycloaddition, has been reported to occur between a radiofluorinated azide and azadibenzocyclooctyne functionalizing the surface of mesoporous silica nanoparticles [144]. Indeed, after the latter were injected and passively accumulated in tumours, they were shown to react with radiofluorinated azido-PEG **44** based on both PET imaging and necropsy data. The fluorination is achieved classically but in this case **44** is injected (2 h after the nanoparticles) and reacts in vivo.

A related approach has been applied for the in vivo radiofluorination of porous silicon nanoparticles [138] pre-targeted with a *trans*-cyclooctene (TCO) moiety. In vivo, the nanoparticle-carried TCO reacted with **78** made of a [¹⁸F]-5-fluoro-5-deoxy-ribose equipped with a tetrazine terminus. The synthesis of **78** is described in Figure 42. In a first step, [¹⁸F]-5-fluoro-5-deoxy-ribose **76** is obtained by a classical tosylate displacement from **75** in the presence of [¹⁸F]-fluoride, K₂CO₃, and K₂.₂.₂, followed by HPLC purification and acidic hydrolysis. In a second step, a tetrazine decorated with an aminoxy function **77** allowed a straightforward oxime linkage with **76** to form **78**. This oxime was then purified again by HPLC. Although the production of the tetrazine derivative appears somehow cumbersome, this approach represents an illustration of the pre-targeting concepts for in vivo labelling of nanoparticles.

To conclude on this overview of the methods involved for the radiolabelling of nanoparticles, as for most radiofluorinations, the smartest approach will consist in application of the "keep it simple" principle. Thus, late radiofluorinations, avoiding tedious purification of any [¹⁸F]-containing intermediates, shall be preferred. In this perspective, one can forecast an increasing use of the radiolabelling techniques based on main group elements such as alumina, silicon, and boron. The radiofluorination of metal-based nanoparticles featuring oxygen atoms might also take advantage of their high thermal stabilities. The generation of such nanoparticles incorporating [¹⁸O]-oxygen atoms can allow the formation of ¹⁸F through the nuclear ¹⁸O(p,n)¹⁸F by direct bombardment of the nanoparticles. Yet in this case the nature of the metal has to be taken into account to avoid the generation of long-lived isotopes through nuclear reaction of the metal itself.
<figure><img src="image_45.png" /><figcaption>**Figure 42.** Synthesis of [¹⁸F]-labelled tetrazine 78 from [¹⁸F]-5-fluoro-5-desoxyribose 75.</figcaption></figure>

**4. Purification Methods and Characterization of Radiolabelled Nanoparticles**

Most articles devoted to radiopharmaceuticals are generally built around three main components: the preparation/synthesis of the compound to be radiolabelled, the radiolabelling method (also including the purification stage), and its biological evaluation (in vitro and/or in vivo). While in the case of a molecular radiocompound, the purification and/or characterization stages of the radioproduct formed are generally provided and detailed (i.e., purification by various chromatography techniques, confirmation of the structure of the radioproduct by HPLC in comparison with its cold analogue, etc.), in the case of radiolabelled nano-objects, the information is often less precise and/or very heterogeneous from one study to another. In other words, in-depth characterization of radiolabelled nanoparticles is generally sorely lacking. This lack of information is also found in the majority of reviews devoted to nanoparticles for SPECT and PET imaging, where no specific item on the purification/characterization of the radioproduct is discussed [22,159–162]. However, there are several recent review articles highlighting the various methods for purifying and characterizing nanoparticles in the field of radiopharmaceuticals/radiolabelled compounds [163–165], confirming the growing need for purification/characterization processes for these radio nano-objects. Readers can refer to recent reviews for further details on the various purification procedures for radiopharmaceuticals and/or the techniques available for the physico-chemical characterization of radiolabelled nanoparticles (including their advantages, disadvantages, and limitations) [163,164].

While the radiolabelling and purification process must be as fast as possible and above all compatible with the short-life of the given radioisotope (110 min for fluorine-18, as mentioned in the introduction), it is clear that the intrinsic nature of the nano-object and the radiolabelling method chosen (direct or indirect) have a strong impact on this process. Due to these radioprobes having a high molecular weight, the purification method of choice is size-exclusion chromatography (SEC) or (ultra)centrifugation and the general use of TLC (or radio-TLC) is the simplest and most widely used technique used to assess the radiochemical purity (RCP) and radiochemical yield (RCY) of radiolabelled nanoparticles. However, alternative purification techniques could be used depending on the nature of the nanoparticles. Thus, considering that the nature of the nanoparticle has more impact on the purification/characterization techniques than the radiolabelling stage, we will categorize the purification stages of various radiofluorinated nano-objects by type of nanoparticles (organic or inorganic platforms as described in the first section). With the help of selected examples, the differences between each process and/or the information provided will
<figure><img src="image_45.png" /><figcaption>**Figure 42.** Synthesis of [¹⁸F]-labelled tetrazine 78 from [¹⁸F]-5-fluoro-5-desoxyribose 75.</figcaption></figure>

**4. Purification Methods and Characterization of Radiolabelled Nanoparticles**

Most articles devoted to radiopharmaceuticals are generally built around three main components: the preparation/synthesis of the compound to be radiolabelled, the radiolabelling method (also including the purification stage), and its biological evaluation (in vitro and/or in vivo). While in the case of a molecular radiocompound, the purification and/or characterization stages of the radioproduct formed are generally provided and detailed (i.e., purification by various chromatography techniques, confirmation of the structure of the radioproduct by HPLC in comparison with its cold analogue, etc.), in the case of radiolabelled nano-objects, the information is often less precise and/or very heterogeneous from one study to another. In other words, in-depth characterization of radiolabelled nanoparticles is generally sorely lacking. This lack of information is also found in the majority of reviews devoted to nanoparticles for SPECT and PET imaging, where no specific item on the purification/characterization of the radioproduct is discussed [22,159–162]. However, there are several recent review articles highlighting the various methods for purifying and characterizing nanoparticles in the field of radiopharmaceuticals/radiolabelled compounds [163–165], confirming the growing need for purification/characterization processes for these radio nano-objects. Readers can refer to recent reviews for further details on the various purification procedures for radiopharmaceuticals and/or the techniques available for the physico-chemical characterization of radiolabelled nanoparticles (including their advantages, disadvantages, and limitations) [163,164].

While the radiolabelling and purification process must be as fast as possible and above all compatible with the short-life of the given radioisotope (110 min for fluorine-18, as mentioned in the introduction), it is clear that the intrinsic nature of the nano-object and the radiolabelling method chosen (direct or indirect) have a strong impact on this process. Due to these radioprobes having a high molecular weight, the purification method of choice is size-exclusion chromatography (SEC) or (ultra)centrifugation and the general use of TLC (or radio-TLC) is the simplest and most widely used technique used to assess the radiochemical purity (RCP) and radiochemical yield (RCY) of radiolabelled nanoparticles. However, alternative purification techniques could be used depending on the nature of the nanoparticles. Thus, considering that the nature of the nanoparticle has more impact on the purification/characterization techniques than the radiolabelling stage, we will categorize the purification stages of various radiofluorinated nano-objects by type of nanoparticles (organic or inorganic platforms as described in the first section). With the help of selected examples, the differences between each process and/or the information provided will
be highlighted, and the characterization of either the radioproduct obtained or the cold
analogue based on fluorine-19 will be discussed.

4.1. *Organic Platforms*

4.1.1. Liposomes, Micelles, and Derivatives

Various [¹⁸F]-liposomal drug delivery systems were studied [27]. Depending on the radiolabelling approach chosen, i.e., surface or intraliposomal radiolabelling (see radiolabelling section), purification methods may vary.

Bioorthogonally labelled [¹⁸F]-liposomes were developed by mixing 3-[^18F]fluoro-1,2-dipalmitoylglycerol ([¹⁸F]-FDPG), as the radioactive tracer, with a lipid formulation [34]. After sonication during 30 min, then filtration through a 0.22 µm filter, the [¹⁸F]-labelled liposomes were suspended in phosphate-buffered saline and obtained with a >95% RCP. More interestingly, the sizes and polydispersity indexes (PDI) of the [¹⁸F]-labelled liposomes and of their cold non-labelled surrogates, measured by DLS, were very close (d = 142.9 nm and PDI = 0.207 for ¹⁸F-compounds vs. d = 130.3 nm and PDI = 0.081 for [¹⁹F]-analogues). In a similar strategy published previously, the [¹⁸F]-FDPG-labelled liposome mixture was sonicated and then extruded through a series of filters (the authors indicated 11 passes through 200 µm diameter pores, then 11 passes through 100 µm diameter pores). It was then purified on gel chromatography (Sephadex G-75, ORTG, Inc. (Oakdale, TN, USA)) using PBS as eluent to remove non-incorporated [¹⁸F]-FDPG [30]. The size distribution of [¹⁸F]-radiolabelled liposomes, measured over one day using a particle size analyzer Nanotrac, was stable over time and comparable to the size of the non-radiolabelled liposomes (mean diameter: 29 ± 9 nm).

An alternative approach called “solid-phase transition method” was used for the one-step [¹⁸F]-labelling of synthesized and pre-formulated liposomes [29]. After incubation of fluorinated amphiphilic intermediates (previously synthesized, purified by semi-preparative HPLC column (C18-column) and obtained with a 100% RCP) with the liposomal solution, the mixture was purified by ultracentrifugation (20 min at 320,000 × g). The efficiency of incorporation of radiofluorinated amphiphilic compounds into liposomes was then determined by HPLC. Interestingly, the authors observed no significant difference in stability between the [¹⁸F]-liposomes and the non-radioactive analogues, confirming the similar structuration of the nano-objects and the possibility of carrying out an initial screening with non-radiolabelled liposomes before developing radiofluorinated analogues of the most promising compounds.

Radiofluorinated liposomes can also be obtained by encapsulating radioactive drugs in the aqueous core of the liposome (intraliposomal radiolabelling). Incorporation of [¹⁸F]-FDG into different negatively, positively, or neutrally charged liposome formulations was achieved by repeated freeze–thaw cycles in liquid nitrogen [166]. The liposomes were extruded and then purified by centrifugation to remove untrapped [¹⁸F]-FDG. Three passes through a 100 nm pore-size polycarbonate filter were required before centrifugating the liposome saline solution (180,000× g for 20 min). A similar process to purify fluorine-labelled dasatinib nanoformulations (micelles and liposomes) was used [14]. Dasatinib was loaded into PEGylated water-soluble nano-carriers. The dasatinib was chemically modified prior to radiofluorination which was performed with no carrier–added [¹⁸F]-KF-Kryptofix® 222complex. After HPLC purification, the pure radioactive intermediate was suspended in NaCl 0.9% and then filtered through a 45 µm filter. It was encapsulated in a liposome (or micelle) using a conventional method. The [¹⁸F]-liposomal formulation was obtained using a Mini Extruder (19 passes through a 100 nm pore-size polycarbonate membrane) and did not require any additional purification. Interestingly, a DLS study of the radiolabelled liposomes (and micelles) was carried out and the loading densities were evaluated. A mean particle size of 120 nm and a drug/lipid molar ratio of 1:2000 were determined for [¹⁸F]-labelled dasatinib liposomes.

Recently, lipid-PEG derivatives with a thiol function or including a peptide bearing a
thiol function were incorporated to sphingomyelin nanometric emulsions (SNs) and then
be highlighted, and the characterization of either the radioproduct obtained or the cold
analogue based on fluorine-19 will be discussed.

4.1. *Organic Platforms*

4.1.1. Liposomes, Micelles, and Derivatives

Various [¹⁸F]-liposomal drug delivery systems were studied [27]. Depending on the radiolabelling approach chosen, i.e., surface or intraliposomal radiolabelling (see radiolabelling section), purification methods may vary.

Bioorthogonally labelled [¹⁸F]-liposomes were developed by mixing 3-[^18F]fluoro-1,2-dipalmitoylglycerol ([¹⁸F]-FDPG), as the radioactive tracer, with a lipid formulation [34]. After sonication during 30 min, then filtration through a 0.22 µm filter, the [¹⁸F]-labelled liposomes were suspended in phosphate-buffered saline and obtained with a >95% RCP. More interestingly, the sizes and polydispersity indexes (PDI) of the [¹⁸F]-labelled liposomes and of their cold non-labelled surrogates, measured by DLS, were very close (d = 142.9 nm and PDI = 0.207 for ¹⁸F-compounds vs. d = 130.3 nm and PDI = 0.081 for [¹⁹F]-analogues). In a similar strategy published previously, the [¹⁸F]-FDPG-labelled liposome mixture was sonicated and then extruded through a series of filters (the authors indicated 11 passes through 200 µm diameter pores, then 11 passes through 100 µm diameter pores). It was then purified on gel chromatography (Sephadex G-75, ORTG, Inc. (Oakdale, TN, USA)) using PBS as eluent to remove non-incorporated [¹⁸F]-FDPG [30]. The size distribution of [¹⁸F]-radiolabelled liposomes, measured over one day using a particle size analyzer Nanotrac, was stable over time and comparable to the size of the non-radiolabelled liposomes (mean diameter: 29 ± 9 nm).

An alternative approach called “solid-phase transition method” was used for the one-step [¹⁸F]-labelling of synthesized and pre-formulated liposomes [29]. After incubation of fluorinated amphiphilic intermediates (previously synthesized, purified by semi-preparative HPLC column (C18-column) and obtained with a 100% RCP) with the liposomal solution, the mixture was purified by ultracentrifugation (20 min at 320,000 × g). The efficiency of incorporation of radiofluorinated amphiphilic compounds into liposomes was then determined by HPLC. Interestingly, the authors observed no significant difference in stability between the [¹⁸F]-liposomes and the non-radioactive analogues, confirming the similar structuration of the nano-objects and the possibility of carrying out an initial screening with non-radiolabelled liposomes before developing radiofluorinated analogues of the most promising compounds.

Radiofluorinated liposomes can also be obtained by encapsulating radioactive drugs in the aqueous core of the liposome (intraliposomal radiolabelling). Incorporation of [¹⁸F]-FDG into different negatively, positively, or neutrally charged liposome formulations was achieved by repeated freeze–thaw cycles in liquid nitrogen [166]. The liposomes were extruded and then purified by centrifugation to remove untrapped [¹⁸F]-FDG. Three passes through a 100 nm pore-size polycarbonate filter were required before centrifugating the liposome saline solution (180,000× g for 20 min). A similar process to purify fluorine-labelled dasatinib nanoformulations (micelles and liposomes) was used [14]. Dasatinib was loaded into PEGylated water-soluble nano-carriers. The dasatinib was chemically modified prior to radiofluorination which was performed with no carrier–added [¹⁸F]-KF-Kryptofix® 222complex. After HPLC purification, the pure radioactive intermediate was suspended in NaCl 0.9% and then filtered through a 45 µm filter. It was encapsulated in a liposome (or micelle) using a conventional method. The [¹⁸F]-liposomal formulation was obtained using a Mini Extruder (19 passes through a 100 nm pore-size polycarbonate membrane) and did not require any additional purification. Interestingly, a DLS study of the radiolabelled liposomes (and micelles) was carried out and the loading densities were evaluated. A mean particle size of 120 nm and a drug/lipid molar ratio of 1:2000 were determined for [¹⁸F]-labelled dasatinib liposomes.

Recently, lipid-PEG derivatives with a thiol function or including a peptide bearing a
thiol function were incorporated to sphingomyelin nanometric emulsions (SNs) and then
radiolabelled with 4-[¹⁸F]-fluorobenzamido-N-ethylmaleimide ([¹⁸F]-FBEM) [12]. Both radiolabelled nanometric emulsions (with or without peptide) were purified by PD-10 desalting column and exhibited hydrodynamic sizes, polydispersity indexes, and ζ potentials (around 140 nm, 0.2 and +38 mV, respectively) similar to those of the non-radioactive analogues, confirming that the radiolabelling approach had no dramatic impact on the nanosystems stability. It should be noted that an extensive HPLC study of the [¹⁸F]-radiolabelled lipid-based nano-carriers degraded in ethanol demonstrated a highly specific radiolabelling through a maleimide-thiol coupling by comparison with the non-radioactive analogue [¹⁹F]-compounds. Interestingly, no trace of the [¹⁸F]-FBEM precursor was observed in any of the chromatograms, thus validating the efficiency of the purification method.

### 4.1.2. Polymers

Most of the [¹⁸F]-radiolabelled polymers were generally purified by (ultra)centrifugation or SEC setup. Radioactive [¹⁸F]-radiolabelled polyester-based nanoparticles were obtained by the nano coprecipitation method using [¹⁸F]-radiolabelled block-copolymers. Whatever the method used to prepare the radiofluorinated nanoparticles, the suspension was purified by centrifugation (4000× g during 45 min at room temperature) with ultracentrifugal filters [45]. The efficiency of the radiolabelling process (and by extension the purification process) was validated by comparing the amount of radioactivity after centrifugation in the precipitate and the supernatant. Surprisingly, the size measurement (diameter < 200 nm, PDI ranging from 0.05 to 0.28) and the ζ potential (around −30 mV) were determined on the radiofluorinated compounds after the complete decay of fluorine-18. The authors concluded that the incorporation of the radioisotope did not modify the physical properties of the nanoparticles.

Modified PLGA nanoparticles were radiolabelled on the basis of the strong non-covalent interaction between avidin and biotin. Specifically, avidin-modified PLGA nanoparticles were [¹⁸F]-labelled with the freshly synthesised biotinylated [¹⁸F]-prosthetic group (purified by semi-preparative HPLC) in a buffer solution. After the radiolabelling step, the efficiency of PLGA radiolabelling was monitored. The resulting radioactive suspension was sonicated, centrifuged (10,000× g for 10 min), and the amount of radioactivity was measured in the filtrate and the precipitate. The amount of radioactivity being at background level in the supernatant, the authors demonstrated that a 5 min sonication of the radiolabelled nanoparticles suspension was sufficient to obtain pure radiolabelled polymer nanoparticles that can be injected into animals [44].

Size-exclusion chromatography, to separate the smaller compounds from the labelled polymer, is an alternative to centrifugation for purifying radiofluorinated polymers. For example, after the [¹⁸F]-labelling of folate-pHPMA conjugates, the radioactive mixture was purified by SEC using three Hitrap desalting columns (Sephadex G-25 resin) in series with 0.9% NaCl solution as solvent [40]. Previously published examples of radiofluorinated HPMA-based polymers also used similar SEC purification. Pure solutions of [¹⁸F]-labelled polymers, ready for in vivo evaluation, were obtained after purification using Hitrap desalting column (Sephadex G-25 Superfine) with 0.9% NaCl solution and a flow rate of 0.5 mL/min [42,167] or with PBS buffer solution and a flow rate of 1 mL/min [41]. Very recently, the radiolabelling of an anionic amphiphilic teroligomer using a CuAAC strategy was studied [168]. Due to the radiolabelling strategy and the anionic nature of the polymer, size-exclusion chromatography (semi-preparative SEC) was performed after a first purification step by solid-phase extraction (to remove the excess of copper salts). The radioactive teroligomer mixture was successively loaded on an anionic exchange cartridge (Chromafix SB cartridge, size M, Macherey-Nagel GmbH & Co., KG, Düren, Germany) and eluted using aqueous 1M HCl solution as eluent, and then the resulting solution was neutralized before being purified using two Hitrap desalting columns in series with phosphate buffer as eluent. Although the polymer was obtained with excellent radiochemical purity and a total reaction time (around 2 h) compatible with the half-life of fluorine-18, the authors indicated a 30% loss of radioactivity during purification by SEC,
radiolabelled with 4-[¹⁸F]-fluorobenzamido-N-ethylmaleimide ([¹⁸F]-FBEM) [12]. Both radiolabelled nanometric emulsions (with or without peptide) were purified by PD-10 desalting column and exhibited hydrodynamic sizes, polydispersity indexes, and ζ potentials (around 140 nm, 0.2 and +38 mV, respectively) similar to those of the non-radioactive analogues, confirming that the radiolabelling approach had no dramatic impact on the nanosystems stability. It should be noted that an extensive HPLC study of the [¹⁸F]-radiolabelled lipid-based nano-carriers degraded in ethanol demonstrated a highly specific radiolabelling through a maleimide-thiol coupling by comparison with the non-radioactive analogue [¹⁹F]-compounds. Interestingly, no trace of the [¹⁸F]-FBEM precursor was observed in any of the chromatograms, thus validating the efficiency of the purification method.

### 4.1.2. Polymers

Most of the [¹⁸F]-radiolabelled polymers were generally purified by (ultra)centrifugation or SEC setup. Radioactive [¹⁸F]-radiolabelled polyester-based nanoparticles were obtained by the nano coprecipitation method using [¹⁸F]-radiolabelled block-copolymers. Whatever the method used to prepare the radiofluorinated nanoparticles, the suspension was purified by centrifugation (4000× g during 45 min at room temperature) with ultracentrifugal filters [45]. The efficiency of the radiolabelling process (and by extension the purification process) was validated by comparing the amount of radioactivity after centrifugation in the precipitate and the supernatant. Surprisingly, the size measurement (diameter < 200 nm, PDI ranging from 0.05 to 0.28) and the ζ potential (around −30 mV) were determined on the radiofluorinated compounds after the complete decay of fluorine-18. The authors concluded that the incorporation of the radioisotope did not modify the physical properties of the nanoparticles.

Modified PLGA nanoparticles were radiolabelled on the basis of the strong non-covalent interaction between avidin and biotin. Specifically, avidin-modified PLGA nanoparticles were [¹⁸F]-labelled with the freshly synthesised biotinylated [¹⁸F]-prosthetic group (purified by semi-preparative HPLC) in a buffer solution. After the radiolabelling step, the efficiency of PLGA radiolabelling was monitored. The resulting radioactive suspension was sonicated, centrifuged (10,000× g for 10 min), and the amount of radioactivity was measured in the filtrate and the precipitate. The amount of radioactivity being at background level in the supernatant, the authors demonstrated that a 5 min sonication of the radiolabelled nanoparticles suspension was sufficient to obtain pure radiolabelled polymer nanoparticles that can be injected into animals [44].

Size-exclusion chromatography, to separate the smaller compounds from the labelled polymer, is an alternative to centrifugation for purifying radiofluorinated polymers. For example, after the [¹⁸F]-labelling of folate-pHPMA conjugates, the radioactive mixture was purified by SEC using three Hitrap desalting columns (Sephadex G-25 resin) in series with 0.9% NaCl solution as solvent [40]. Previously published examples of radiofluorinated HPMA-based polymers also used similar SEC purification. Pure solutions of [¹⁸F]-labelled polymers, ready for in vivo evaluation, were obtained after purification using Hitrap desalting column (Sephadex G-25 Superfine) with 0.9% NaCl solution and a flow rate of 0.5 mL/min [42,167] or with PBS buffer solution and a flow rate of 1 mL/min [41]. Very recently, the radiolabelling of an anionic amphiphilic teroligomer using a CuAAC strategy was studied [168]. Due to the radiolabelling strategy and the anionic nature of the polymer, size-exclusion chromatography (semi-preparative SEC) was performed after a first purification step by solid-phase extraction (to remove the excess of copper salts). The radioactive teroligomer mixture was successively loaded on an anionic exchange cartridge (Chromafix SB cartridge, size M, Macherey-Nagel GmbH & Co., KG, Düren, Germany) and eluted using aqueous 1M HCl solution as eluent, and then the resulting solution was neutralized before being purified using two Hitrap desalting columns in series with phosphate buffer as eluent. Although the polymer was obtained with excellent radiochemical purity and a total reaction time (around 2 h) compatible with the half-life of fluorine-18, the authors indicated a 30% loss of radioactivity during purification by SEC,
which explains the rather low radiochemical yield (around 15%). It should be noted that no information was provided by the authors on any loss of radioactivity during the initial purification by solid phase extraction.

In the case of the first tumour-targeting nanoparticles reported with fluorine-18, the purification step was a little different [50]. Using selected amphiphilic block-copolymers, nanoparticles and micelles were characterized by DLS (in solution) prior fluorination (hydrodynamic diameters from 47 to 142 nm). After reaction with [¹⁸F]-fluoride in the presence of Kryptofix® 222and K₂CO₃, the radiofluorinated nanoparticles were diluted with water and passed through a column containing Dowex strongly acidic ion exchange resin, followed by a short plug of alumina. These conditions removed all of the Kryptofix® 222 and most of the unreacted fluoride ion. The radiochemical purity measured by radio-TLC was 61% (i.e., 31% of the fluoride was incorporated into the nanoparticles). No control of the size after labelling was performed.

### 4.1.3. Dendrimers

A few examples of radiofluorinated dendrimers have been reported as functionalized PAMAM derivatives (half dendrimers) [53]. Aryltrifluoroborates functionalized dendron-biotin conjugates (4, 6, and 16 branch dendrons) were radiolabelled with [¹⁸F]-FF⁻ before the resulting solution was purified via a silica cartridge using an acetonitrile/water mixture as eluent. The purification step was monitored using TLC (ACN/water 7/3) and no trace of free or unreacted [¹⁸F]-fluoride was detected in the solution. PAMAM (G6) dendrimer derivatives [54], radiolabelled via a [¹⁸F]-prosthetic group, were purified using a Sephadex G-25 column. The radiochemical purity was then checked using analytical HPLC (C18-column, eluent: water 0.1% TFA/ACN). More recently, various works based on the radiosynthesis and preclinical evaluation of a G4 PAMAM hydroxyl dendrimer (called OP-801) as a [¹⁸F]-PET tracer were reported [55,56]. A click chemistry strategy (CuAAC bioconjugation) was used to radiofluorinate the dendrimer, named [¹⁸F]-OP-801. It was first purified by semi-preparative HPLC (Phenomenex Biosec-SEC-s-2000) and the resulting solution was then passed through a Sep-Pak C18 plus cartridge (washed with sterile water before elution using dehydrated ethanol (1 mL) and then 0.9% sodium chloride solution (10 mL)). The RCP was >95% and the purity of the radiocompound was checked by analytical HPLC (Phenomenex Jupiter 5 mm C18). More interestingly, the radiosynthesis and purification steps were optimized and automated using a purification procedure identical to that mentioned above (HPLC followed by trapping on a Sep-Pak C18). Clinical grade [¹⁸F]-OP-801 suitable for human use was thus produced under "Good Manufacturing Practices" conditions, and enhanced quality control was implemented to meet the criteria of the food and drug administrations. Thus, several batches of radioactive compounds were prepared, purified, analysed, and identified. A calibration curve (developed with the cold analogue [¹⁹F]-OP-801) was used to determine the total carrier mass of non-radioactive [¹⁸F]-OP-801 and derivatives. The structure of [¹⁸F]-OP-801 was confirmed by a comparative HPLC study (similar retention times for [¹⁸F]-OP-801 compared with [¹⁹F]-OP-801).

### 4.1.4. Nanodroplets

Perfluorocabon (PFC) nanodroplets were radiolabelled by adding a radiofluorinated monomer ([¹⁸F]-CF₃(CF₂)₇(CH₂)₃F) in the presence of an emulsifier (perfluorohexane with a fluorosurfactant (Zonyl-FSO) or perfluorooctylbromide with a lipid solution) [10]. The mixture was sonicated for 10 min, and then centrifuged for 20 min at 4000 rpm. The resulting precipitate was redispersed in distilled water to a volume concentration of 6% radioactive droplets. The [¹⁸F]-PFC droplets were stable (loss of [¹⁸F]-F⁻ below 2.4% after 2 h) and the determination of their classical properties (hydrodynamic diameters and ζ potentials) showed no change compared to their non-radioactive analogues.
which explains the rather low radiochemical yield (around 15%). It should be noted that no information was provided by the authors on any loss of radioactivity during the initial purification by solid phase extraction.

In the case of the first tumour-targeting nanoparticles reported with fluorine-18, the purification step was a little different [50]. Using selected amphiphilic block-copolymers, nanoparticles and micelles were characterized by DLS (in solution) prior fluorination (hydrodynamic diameters from 47 to 142 nm). After reaction with [¹⁸F]-fluoride in the presence of Kryptofix® 222and K₂CO₃, the radiofluorinated nanoparticles were diluted with water and passed through a column containing Dowex strongly acidic ion exchange resin, followed by a short plug of alumina. These conditions removed all of the Kryptofix® 222 and most of the unreacted fluoride ion. The radiochemical purity measured by radio-TLC was 61% (i.e., 31% of the fluoride was incorporated into the nanoparticles). No control of the size after labelling was performed.

### 4.1.3. Dendrimers

A few examples of radiofluorinated dendrimers have been reported as functionalized PAMAM derivatives (half dendrimers) [53]. Aryltrifluoroborates functionalized dendron-biotin conjugates (4, 6, and 16 branch dendrons) were radiolabelled with [¹⁸F]-FF⁻ before the resulting solution was purified via a silica cartridge using an acetonitrile/water mixture as eluent. The purification step was monitored using TLC (ACN/water 7/3) and no trace of free or unreacted [¹⁸F]-fluoride was detected in the solution. PAMAM (G6) dendrimer derivatives [54], radiolabelled via a [¹⁸F]-prosthetic group, were purified using a Sephadex G-25 column. The radiochemical purity was then checked using analytical HPLC (C18-column, eluent: water 0.1% TFA/ACN). More recently, various works based on the radiosynthesis and preclinical evaluation of a G4 PAMAM hydroxyl dendrimer (called OP-801) as a [¹⁸F]-PET tracer were reported [55,56]. A click chemistry strategy (CuAAC bioconjugation) was used to radiofluorinate the dendrimer, named [¹⁸F]-OP-801. It was first purified by semi-preparative HPLC (Phenomenex Biosec-SEC-s-2000) and the resulting solution was then passed through a Sep-Pak C18 plus cartridge (washed with sterile water before elution using dehydrated ethanol (1 mL) and then 0.9% sodium chloride solution (10 mL)). The RCP was >95% and the purity of the radiocompound was checked by analytical HPLC (Phenomenex Jupiter 5 mm C18). More interestingly, the radiosynthesis and purification steps were optimized and automated using a purification procedure identical to that mentioned above (HPLC followed by trapping on a Sep-Pak C18). Clinical grade [¹⁸F]-OP-801 suitable for human use was thus produced under "Good Manufacturing Practices" conditions, and enhanced quality control was implemented to meet the criteria of the food and drug administrations. Thus, several batches of radioactive compounds were prepared, purified, analysed, and identified. A calibration curve (developed with the cold analogue [¹⁹F]-OP-801) was used to determine the total carrier mass of non-radioactive [¹⁸F]-OP-801 and derivatives. The structure of [¹⁸F]-OP-801 was confirmed by a comparative HPLC study (similar retention times for [¹⁸F]-OP-801 compared with [¹⁹F]-OP-801).

### 4.1.4. Nanodroplets

Perfluorocabon (PFC) nanodroplets were radiolabelled by adding a radiofluorinated monomer ([¹⁸F]-CF₃(CF₂)₇(CH₂)₃F) in the presence of an emulsifier (perfluorohexane with a fluorosurfactant (Zonyl-FSO) or perfluorooctylbromide with a lipid solution) [10]. The mixture was sonicated for 10 min, and then centrifuged for 20 min at 4000 rpm. The resulting precipitate was redispersed in distilled water to a volume concentration of 6% radioactive droplets. The [¹⁸F]-PFC droplets were stable (loss of [¹⁸F]-F⁻ below 2.4% after 2 h) and the determination of their classical properties (hydrodynamic diameters and ζ potentials) showed no change compared to their non-radioactive analogues.
## 4.2. Inorganic Platforms

Although the nanoparticle size is also the main criterion for the purification steps of radiofluorinated inorganic nanoparticles, depending on their nature (e.g., magnetic nanoparticles) and composition, other purification and/or characterization techniques shall be used, such as X-ray diffraction (XRD) or X-ray photoelectron spectroscopy (XPS) for the determination of structure/crystallinity or surface speciation, respectively.

### 4.2.1. Rare Earth Nanoparticles

Numerous examples of radiofluorination of rare earth nanoparticles (REs) have been reported over the last decade [58,59,62,66,69,70,84]. As said above, this growing interest is based on the strong binding between $[^{18}F]-F^-$ and the rare earth cations as well as on the unique 4f electronic structure of rare earth ions, which gives them peculiar optical and/or magnetic properties, and could therefore lead to bi- or trimodal (optical and/or magnetic and nuclear) imaging nanoprobes.

Whatever the system studied (naked REs or coated REs or REs functionalized by a biological vector), the nanoparticles (and their eventual surface coating) were characterized before radiofluorination using the standard analytical techniques (TEM, XRD, XPS, IR, TGA, etc.). Then, radiolabelling was carried out by incubating the REs with $[^{18}F]-FF^-$ (from 1 to 10 min at room temperature) and the purification process consisted of a first centrifugation step followed by several water washes with sonication and centrifugation to remove the unreacted $[^{18}F]-F^-$. Radiopurity was monitored by radio-TLC. It is suggested that long-time and high-speed centrifugation of ultrasmall REs during the purification stage could induce aggregation of the nanoparticles. This hypothesis was put forward to explain the high uptake of radiolabelled sub-5 nm $KGdF_4$ REs in the lungs (45% ID/g) [59].

Interestingly, in one case, a model study of the radiolabelling/purification process with cold nanoparticles ($[^{19}F]$-material) was reported [58]. To establish the reaction between fluoride ions and RE nanoparticles, tetra-*n*-butylammonium fluoride (as a source of $[^{19}F]-F^-$) was incubated with F127-modified $Y_2O_3$ nanoparticles for 1 min at room temperature in $CHCl_3$ before purification (centrifugation + washings), and the incorporation of $FF^-$ was investigated by XPS. In contrast to the starting material, the $F_{1s}$ peak at 685.6 eV was observed after $^{19}F$ incorporation (Figure 43b, XPS spectra). Using the same protocol, $[^{18}F]$-radiolabelled REs were easily obtained, as shown in the Figure 43a, autoradiography, with no trace of residual $[^{18}F]-F^-$ detected, thus validating the purification process.

<figure><img src="image_46.png" /><figcaption>**Figure 43.** $^{18/19}F$-REs characterization. (a) $[^{18}F]$-labelling monitory of REs (autoradiography performed in saline buffer; (b) rare earth nanoparticles before and after incorporation of a fluorine source (tetra-*n*-butylammonium fluoride) in $CHCl_3$ at ambient temperature and purification by washing and centrifugation (reprinted from Biomaterials, 32, Sun, Y.; Yu, M.; Liang, S.; Zhang, Y.; Li, C.; Mou, T.; Yang, W.; Zhang, X.; Li, B.; Huang, C.; Li, F. Fluorine-18-labeled rare earth nanoparticles for positron emission tomography (PET) imaging of sentinel lymph node, 2999–3007, Copyright 2011, with the permission from Elsevier).</figcaption></figure>
## 4.2. Inorganic Platforms

Although the nanoparticle size is also the main criterion for the purification steps of radiofluorinated inorganic nanoparticles, depending on their nature (e.g., magnetic nanoparticles) and composition, other purification and/or characterization techniques shall be used, such as X-ray diffraction (XRD) or X-ray photoelectron spectroscopy (XPS) for the determination of structure/crystallinity or surface speciation, respectively.

### 4.2.1. Rare Earth Nanoparticles

Numerous examples of radiofluorination of rare earth nanoparticles (REs) have been reported over the last decade [58,59,62,66,69,70,84]. As said above, this growing interest is based on the strong binding between $[^{18}F]-F^-$ and the rare earth cations as well as on the unique 4f electronic structure of rare earth ions, which gives them peculiar optical and/or magnetic properties, and could therefore lead to bi- or trimodal (optical and/or magnetic and nuclear) imaging nanoprobes.

Whatever the system studied (naked REs or coated REs or REs functionalized by a biological vector), the nanoparticles (and their eventual surface coating) were characterized before radiofluorination using the standard analytical techniques (TEM, XRD, XPS, IR, TGA, etc.). Then, radiolabelling was carried out by incubating the REs with $[^{18}F]-FF^-$ (from 1 to 10 min at room temperature) and the purification process consisted of a first centrifugation step followed by several water washes with sonication and centrifugation to remove the unreacted $[^{18}F]-F^-$. Radiopurity was monitored by radio-TLC. It is suggested that long-time and high-speed centrifugation of ultrasmall REs during the purification stage could induce aggregation of the nanoparticles. This hypothesis was put forward to explain the high uptake of radiolabelled sub-5 nm $KGdF_4$ REs in the lungs (45% ID/g) [59].

Interestingly, in one case, a model study of the radiolabelling/purification process with cold nanoparticles ($[^{19}F]$-material) was reported [58]. To establish the reaction between fluoride ions and RE nanoparticles, tetra-*n*-butylammonium fluoride (as a source of $[^{19}F]-F^-$) was incubated with F127-modified $Y_2O_3$ nanoparticles for 1 min at room temperature in $CHCl_3$ before purification (centrifugation + washings), and the incorporation of $FF^-$ was investigated by XPS. In contrast to the starting material, the $F_{1s}$ peak at 685.6 eV was observed after $^{19}F$ incorporation (Figure 43b, XPS spectra). Using the same protocol, $[^{18}F]$-radiolabelled REs were easily obtained, as shown in the Figure 43a, autoradiography, with no trace of residual $[^{18}F]-F^-$ detected, thus validating the purification process.

<figure><img src="image_46.png" /><figcaption>**Figure 43.** $^{18/19}F$-REs characterization. (a) $[^{18}F]$-labelling monitory of REs (autoradiography performed in saline buffer; (b) rare earth nanoparticles before and after incorporation of a fluorine source (tetra-*n*-butylammonium fluoride) in $CHCl_3$ at ambient temperature and purification by washing and centrifugation (reprinted from Biomaterials, 32, Sun, Y.; Yu, M.; Liang, S.; Zhang, Y.; Li, C.; Mou, T.; Yang, W.; Zhang, X.; Li, B.; Huang, C.; Li, F. Fluorine-18-labeled rare earth nanoparticles for positron emission tomography (PET) imaging of sentinel lymph node, 2999–3007, Copyright 2011, with the permission from Elsevier).</figcaption></figure>
### 4.2.2. Metal Oxides

One example of ceria (CeO₂) nanoparticles [¹⁸F]-radiolabelling was reported in 2012 [89]. The amino groups of the functionalized CeO₂ nanoparticles were incubated with [¹⁸F]-SFB, 1 h at room temperature. The radioactive mixture was purified by successive centrifugation/washing cycles until no radioactivity was detected in the supernatant (4000 rpm for 10 min/PBS). Pure [¹⁸F]-CeO₂ nanoparticles were suspended in PBS/tween 80 and sonicated 5 min prior to in vivo injection. Structural analysis (elemental analysis, thermogravimetry, and IR spectroscopy) of CeO₂ nanoparticles was carried out before the radiolabelling to demonstrate their successful functionalization and the presence of 3-aminopropyl groups anchored on the ceria surface (the density of aminopropyl groups was approximately 4.4 mmol g⁻¹).

Various works described [¹⁸F]-radiolabelling of metal oxides using the click chemistry strategy such as that on [¹⁸F]-nanoparticles based on cross-linked dextran iron oxide ([¹⁸F]-CLIO) [74]. In this work, the [¹⁸F]-propargyl PEG₃ synthon was readily clicked onto azido-PEG-modified CLIO in PBS buffer. Microcentrifugation filtration of the resulting mixture yielded pure [¹⁸F]-CLIO. The radiochemical purity determined by analytical HPLC (C18 column) was >98%. More recently, RGD peptide and fluorine-18 were coupled onto ultrasmall iron oxide nanoparticles (USPIO) by click chemistry [78]. The resulting solution was purified by ultrafiltration using 100kDa molecular weight cut-off filters to remove the unreacted [¹⁸F]-prosthetic moiety ([¹⁸F]-FEA). Filtration was carried out until the amount of radioactivity was at background level in the filtrate. The nanoparticle solution was then concentrated before being used. The radiolabelling and purification process were completed in just 1 h. The first in vivo biodistribution of [¹⁸F]-RGD@USPIO revealed a high uptake in bones that was attributed by the authors to the incomplete elimination of unreacted [¹⁸F]-FEA during the purification process. However, whether this bone uptake could be linked to an in vivo defluorination reaction is questionable. It is noteworthy that, in this case, the isotopologue [¹⁹F]-RGD@USPIO was prepared in the same conditions and its physico-chemical features determined: core size of 6.5 ± 0.5 nm and average hydrodynamic diameter of 40.44 nm.

Unlike for molecular species, HPLC is rarely used to purify inorganic nanoparticles due to uncontrolled aggregation that could definitely clot the capillaries. However, modification of a HPLC system was recently reported as an innovative strategy for the purification of [¹⁸F]-radiolabelled Fe₃O₄ nanoparticles [79]. The HPLC system was modified by removing the column, and coiling the capillary tube, through which the solution circulates, around a magnet (Figure 44). Thus, during purification of the solution, the non-magnetic particles (unreacted materials, side-products, etc.) are rapidly eliminated during elution/washing of the solution, while the radiolabelled nanoparticles are retained by the magnet. Once the magnet is removed, the pure nanoparticles can be collected. Interestingly, this magnetic separation process could be extended to other radiolabelled magnetic nanoparticles.

This strategy was applied to purify radiolabelled aluminium hydroxide-coated magnetite nanoparticles with [¹⁸F]-F⁻. Indeed, after Fe₃O₄@Al(OH)₃ and [¹⁸F]-F⁻ were incubated for 10–15 min at room temperature in water, the solution was centrifugated at 3000–4000 rpm for 20 min and the residue washed several times with water until no radioactivity was collected in the filtrate, before being re-suspended in saline solution. It was further shown that magnetic separation using a strong permanent magnet (NdFeB magnet) could replace the purification by centrifugation. It should be noted that the radiolabelling and purification steps were validated upstream with [¹⁹F]-nanoparticles [82,83].

An alternative route towards [¹⁸F]-labelled water-soluble functionalized iron oxide (IO) nanoparticles is to stabilize [¹⁸F]-aluminium fluoride ([¹⁸F]-AlF) using a chelator such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) [80]. [¹⁸F]-AlF labelling and [¹⁹F]-AlF chelation of NOTA-IO nanoparticles were performed. In both cases, the desired product (([¹⁸/¹⁹F]-AlF)-NOTA-IO nanoparticles) was purified using a size-exclusion column (PD-10) with PBS as eluent then concentrated with a centrifugal filter (MWCO = 30 kDa). The number of AlF units per nanoparticle determined by ICP-MS, at different pH values, ranged
### 4.2.2. Metal Oxides

One example of ceria (CeO₂) nanoparticles [¹⁸F]-radiolabelling was reported in 2012 [89]. The amino groups of the functionalized CeO₂ nanoparticles were incubated with [¹⁸F]-SFB, 1 h at room temperature. The radioactive mixture was purified by successive centrifugation/washing cycles until no radioactivity was detected in the supernatant (4000 rpm for 10 min/PBS). Pure [¹⁸F]-CeO₂ nanoparticles were suspended in PBS/tween 80 and sonicated 5 min prior to in vivo injection. Structural analysis (elemental analysis, thermogravimetry, and IR spectroscopy) of CeO₂ nanoparticles was carried out before the radiolabelling to demonstrate their successful functionalization and the presence of 3-aminopropyl groups anchored on the ceria surface (the density of aminopropyl groups was approximately 4.4 mmol g⁻¹).

Various works described [¹⁸F]-radiolabelling of metal oxides using the click chemistry strategy such as that on [¹⁸F]-nanoparticles based on cross-linked dextran iron oxide ([¹⁸F]-CLIO) [74]. In this work, the [¹⁸F]-propargyl PEG₃ synthon was readily clicked onto azido-PEG-modified CLIO in PBS buffer. Microcentrifugation filtration of the resulting mixture yielded pure [¹⁸F]-CLIO. The radiochemical purity determined by analytical HPLC (C18 column) was >98%. More recently, RGD peptide and fluorine-18 were coupled onto ultrasmall iron oxide nanoparticles (USPIO) by click chemistry [78]. The resulting solution was purified by ultrafiltration using 100kDa molecular weight cut-off filters to remove the unreacted [¹⁸F]-prosthetic moiety ([¹⁸F]-FEA). Filtration was carried out until the amount of radioactivity was at background level in the filtrate. The nanoparticle solution was then concentrated before being used. The radiolabelling and purification process were completed in just 1 h. The first in vivo biodistribution of [¹⁸F]-RGD@USPIO revealed a high uptake in bones that was attributed by the authors to the incomplete elimination of unreacted [¹⁸F]-FEA during the purification process. However, whether this bone uptake could be linked to an in vivo defluorination reaction is questionable. It is noteworthy that, in this case, the isotopologue [¹⁹F]-RGD@USPIO was prepared in the same conditions and its physico-chemical features determined: core size of 6.5 ± 0.5 nm and average hydrodynamic diameter of 40.44 nm.

Unlike for molecular species, HPLC is rarely used to purify inorganic nanoparticles due to uncontrolled aggregation that could definitely clot the capillaries. However, modification of a HPLC system was recently reported as an innovative strategy for the purification of [¹⁸F]-radiolabelled Fe₃O₄ nanoparticles [79]. The HPLC system was modified by removing the column, and coiling the capillary tube, through which the solution circulates, around a magnet (Figure 44). Thus, during purification of the solution, the non-magnetic particles (unreacted materials, side-products, etc.) are rapidly eliminated during elution/washing of the solution, while the radiolabelled nanoparticles are retained by the magnet. Once the magnet is removed, the pure nanoparticles can be collected. Interestingly, this magnetic separation process could be extended to other radiolabelled magnetic nanoparticles.

This strategy was applied to purify radiolabelled aluminium hydroxide-coated magnetite nanoparticles with [¹⁸F]-F⁻. Indeed, after Fe₃O₄@Al(OH)₃ and [¹⁸F]-F⁻ were incubated for 10–15 min at room temperature in water, the solution was centrifugated at 3000–4000 rpm for 20 min and the residue washed several times with water until no radioactivity was collected in the filtrate, before being re-suspended in saline solution. It was further shown that magnetic separation using a strong permanent magnet (NdFeB magnet) could replace the purification by centrifugation. It should be noted that the radiolabelling and purification steps were validated upstream with [¹⁹F]-nanoparticles [82,83].

An alternative route towards [¹⁸F]-labelled water-soluble functionalized iron oxide (IO) nanoparticles is to stabilize [¹⁸F]-aluminium fluoride ([¹⁸F]-AlF) using a chelator such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) [80]. [¹⁸F]-AlF labelling and [¹⁹F]-AlF chelation of NOTA-IO nanoparticles were performed. In both cases, the desired product (([¹⁸/¹⁹F]-AlF)-NOTA-IO nanoparticles) was purified using a size-exclusion column (PD-10) with PBS as eluent then concentrated with a centrifugal filter (MWCO = 30 kDa). The number of AlF units per nanoparticle determined by ICP-MS, at different pH values, ranged
from 1243 ± 294 at pH = 4 to 624 ± 137 at pH = 4.5, this last value (and consequently the associated radioactivity) being sufficient for the authors to carry out radiolabelling at pH 4.5.

<figure><img src="image_47.png" /></figure>

**Figure 44.** Magnetic separation strategy: schematic construction of the HPLC system including a magnet (Used with permission of Biomaterials Science, from ¹⁸F-labeled magnetic nanovectors for bimodal cellular imaging, Schütz, M.B.; Renner, A.M.; Ilyas, S.; Lê, K.; Guliyev, M.; Krapf, P.; Neumaier, B.; Mathur, S., 9, 2021, Copyright 2013, permission conveyed through Copyright Clearance Center, Inc.).

Syntheses of [¹⁸F]-labelled metal oxide nanoparticles by direct proton beam irradiation of ¹⁸O-enriched aluminium oxide or ¹⁸O-enriched titanium oxide were also studied [85,90]. For example, after irradiating ¹⁸O-enriched aluminium oxide with 16 MeV protons, the radioactive nanoparticles were stored until the ¹³N (produced during the proton irradiation) had completely decayed (t₁/₂ = 9.97 min). The radioactive nanoparticles were suspended in NaCl solution, sonicated for 2 min then centrifuged at 8000 × g before being used. This elegant radiolabelling method requires no real purification and has no dramatic impact on the main features of the nanoparticles, as XRD and DLS measurements taken before and after irradiation showed no relevant changes.

The last example of radiofluoration of metal oxide nanoparticles concerns zinc oxide nanoparticles [88]. [¹⁸F]-ZnO nanoparticles were obtained by CuAAC reaction. The radioactive nanoparticles were purified by centrifugation (10,000 rpm for 5 min) and the precipitate was washed with HEPES buffer three times prior being resuspended in HEPES buffer. The radiochemical purity determined by TLC was >95%. In vivo experiments confirmed the efficiency of the purification step, as no bone uptake was observed. So, no unreacted [¹⁸F]-F⁻ or [¹⁸F]-PEGazide **44** (see Figure 29) intermediate were present in the injected [¹⁸F]-ZnO nanoparticles solution.

### 4.2.3. Gold Nanoparticles

While gold nanoparticles have been widely used in biological applications [169], the first biological evaluation of [¹⁸F]-labelled gold nanoparticles was published in 2012 [120]. They were obtained by conjugation reaction between [¹⁸F]-SFB **38** (see Figure 25) and the amino group of the lysine residues of the peptide (peptide CK) anchored to the nanoparticles. The crude solution was centrifugated at 4200 rpm (ca. 1980 g) for 15 min and the resulting precipitate was washed three times until no amount of radioactivity was detected in the supernatant, then the solid was suspended in sodium citrate/tween80 mixture. The nanoparticles were fully characterized by DLS, XPS and UV-Vis prior [¹⁸F]-labelling.
from 1243 ± 294 at pH = 4 to 624 ± 137 at pH = 4.5, this last value (and consequently the associated radioactivity) being sufficient for the authors to carry out radiolabelling at pH 4.5.

<figure><img src="image_47.png" /></figure>

**Figure 44.** Magnetic separation strategy: schematic construction of the HPLC system including a magnet (Used with permission of Biomaterials Science, from ¹⁸F-labeled magnetic nanovectors for bimodal cellular imaging, Schütz, M.B.; Renner, A.M.; Ilyas, S.; Lê, K.; Guliyev, M.; Krapf, P.; Neumaier, B.; Mathur, S., 9, 2021, Copyright 2013, permission conveyed through Copyright Clearance Center, Inc.).

Syntheses of [¹⁸F]-labelled metal oxide nanoparticles by direct proton beam irradiation of ¹⁸O-enriched aluminium oxide or ¹⁸O-enriched titanium oxide were also studied [85,90]. For example, after irradiating ¹⁸O-enriched aluminium oxide with 16 MeV protons, the radioactive nanoparticles were stored until the ¹³N (produced during the proton irradiation) had completely decayed (t₁/₂ = 9.97 min). The radioactive nanoparticles were suspended in NaCl solution, sonicated for 2 min then centrifuged at 8000 × g before being used. This elegant radiolabelling method requires no real purification and has no dramatic impact on the main features of the nanoparticles, as XRD and DLS measurements taken before and after irradiation showed no relevant changes.

The last example of radiofluorination of metal oxide nanoparticles concerns zinc oxide nanoparticles [88]. [¹⁸F]-ZnO nanoparticles were obtained by CuAAC reaction. The radioactive nanoparticles were purified by centrifugation (10,000 rpm for 5 min) and the precipitate was washed with HEPES buffer three times prior being resuspended in HEPES buffer. The radiochemical purity determined by TLC was >95%. In vivo experiments confirmed the efficiency of the purification step, as no bone uptake was observed. So, no unreacted [¹⁸F]-F⁻ or [¹⁸F]-PEGazide **44** (see Figure 29) intermediate were present in the injected [¹⁸F]-ZnO nanoparticles solution.

### 4.2.3. Gold Nanoparticles

While gold nanoparticles have been widely used in biological applications [169], the first biological evaluation of [¹⁸F]-labelled gold nanoparticles was published in 2012 [120]. They were obtained by conjugation reaction between [¹⁸F]-SFB **38** (see Figure 25) and the amino group of the lysine residues of the peptide (peptide CK) anchored to the nanoparticles. The crude solution was centrifugated at 4200 rpm (ca. 1980 g) for 15 min and the resulting precipitate was washed three times until no amount of radioactivity was detected in the supernatant, then the solid was suspended in sodium citrate/tween80 mixture. The nanoparticles were fully characterized by DLS, XPS and UV-Vis prior [¹⁸F]-labelling.
Based on the radioactivity/mass ratio, an average of 27 [¹⁸F]-atoms per nanoparticle was determined. In addition, stability studies were carried out with [¹⁹F]-gold nanoparticles and with [¹⁸F]-labelled gold nanoparticles, according to UV-Vis spectra. [¹⁸F]-labelled nanoparticles were stable for at least 5 h in PBS solution.

After rapid production of [¹⁸F]-PEGylated gold nanoparticles using SiFA radiolabelling strategy [122], size-exclusion gel purification (NAP-10 column with 0.1M sodium buffer as eluent) yielded pure [¹⁸F]-labelled gold nanoparticles. The purity was confirmed by radio-TLC (R<sub>f</sub> = 0 for all [¹⁸F]-labelled nanoparticles). Based on stoichiometry, only three silicon-fluorine prosthetic groups [¹⁸F]-SiFA-SH (and therefore three fluorine-18) were grafted onto each maleimide-gold nanoparticle. The XPS data provided additional proof of successful Michael addition reaction by the appearance of a new peak related to the fluorine at 686.8 eV (corresponding to F<sub>1s</sub>) for the radiofluorinated gold nanoparticles (see Figure 45). As indicated by the authors, the radioactive nanoparticles were stored in a shielded container for 24 h until they had decayed sufficiently to be analysed by XPS. Given the decay of fluorine-18 over 24 h, one can consider that the fluorine peak observed by XPS is due to [¹⁹F]-nanoparticles and not to [¹⁸F]-nanoparticles.

<figure><img src="image_48.png" /><figcaption>**Figure 45.** XPS spectra proving the [¹⁸F]-radiolabelling of gold nanoparticles. (a) [¹⁸F]-SiFA-maleimide-nanoparticles; (b) maleimide-nanoparticles; (c) offset high resolution scans of F1s of [¹⁸F]-</figcaption></figure>
Based on the radioactivity/mass ratio, an average of 27 [¹⁸F]-atoms per nanoparticle was determined. In addition, stability studies were carried out with [¹⁹F]-gold nanoparticles and with [¹⁸F]-labelled gold nanoparticles, according to UV-Vis spectra. [¹⁸F]-labelled nanoparticles were stable for at least 5 h in PBS solution.

After rapid production of [¹⁸F]-PEGylated gold nanoparticles using SiFA radiolabelling strategy [122], size-exclusion gel purification (NAP-10 column with 0.1M sodium buffer as eluent) yielded pure [¹⁸F]-labelled gold nanoparticles. The purity was confirmed by radio-TLC (R<sub>f</sub> = 0 for all [¹⁸F]-labelled nanoparticles). Based on stoichiometry, only three silicon-fluorine prosthetic groups [¹⁸F]-SiFA-SH (and therefore three fluorine-18) were grafted onto each maleimide-gold nanoparticle. The XPS data provided additional proof of successful Michael addition reaction by the appearance of a new peak related to the fluorine at 686.8 eV (corresponding to F<sub>1s</sub>) for the radiofluorinated gold nanoparticles (see Figure 45). As indicated by the authors, the radioactive nanoparticles were stored in a shielded container for 24 h until they had decayed sufficiently to be analysed by XPS. Given the decay of fluorine-18 over 24 h, one can consider that the fluorine peak observed by XPS is due to [¹⁹F]-nanoparticles and not to [¹⁸F]-nanoparticles.

<figure><img src="image_48.png" /><figcaption>**Figure 45.** XPS spectra proving the [¹⁸F]-radiolabelling of gold nanoparticles. (a) [¹⁸F]-SiFA-maleimide-nanoparticles; (b) maleimide-nanoparticles; (c) offset high resolution scans of F1s of [¹⁸F]-</figcaption></figure>
SiFA-maleimide-nanoparticles (green) and maleimide-nanoparticles (red) (analysis carried out with partially decayed [¹⁸F]-SiFA-maleimide-nanoparticles) (Reprinted with permission from Rapid ¹⁸F-labeling and loading of PEGylated gold nanoparticles for in vivo applications, Zhu, J.; Chin, J.; Wängler, C.; Wängler, B.; Lennox, R.B.; Schirrmacher, R., Bioconjugate Chemistry 2014, 25, 1143–1150, Copyright 2014, American Chemical Society).

Similar work in terms of purification and characterization was focused on the [¹⁸F]-radiolabelling of nitrone-gold nanoparticles using a bioorthogonal strain-promoted alkyne-nitrone cycloaddition (SPANC reaction) [124]. The radiofluorinated nanoparticles obtained from the SPANC reaction were passed through a PD-10 desalting column using 0.1M PBS buffer as eluent, leading to a pure [¹⁸F]-labelled gold nanoparticles solution ready to use. An extensive structural study (¹H NMR, IR, TEM and thermogravimetric analysis (TGA)) was carried out on non-radioactive nanoparticles (nitrone-gold nanoparticles and [¹⁹F]-labelled gold nanoparticles). Briefly, the nitrone-gold nanoparticles were spherical with a gold core of 2.6 ± 0.5 nm wide and perfectly monodisperse. Remarkably, the authors determined the raw formula of the nanoparticles (Au₅₀₀(MeO−EG₃−S⁻)₂₀₀(nitrone−EG₄−S⁻)₄₀, EG = ethylene glycol) as well as the amount of nitrone moieties per nanoparticle (0.840 µmol.mg⁻¹). The feasibility of the SPANC reaction was mainly demonstrated by NMR and XPS analyses on the isotopologous ¹⁹F model. The ¹⁹F NMR spectrum of [¹⁹F]-labelled gold nanoparticle recorded in D₂O showed only one peak at −222.91 ppm and comparison of the XPS spectra before and after conjugation exhibited the appearance of new peaks at 289.2 and 686.7 eV corresponding to C₁s and F₁s (C-F bond) for the [¹⁹F]-labelled gold nanoparticle, respectively. An average of 40 ¹⁹F atoms per nanoparticle were determined.

Finally, a less conventional nanoparticle architecture in which a [¹⁸F]-FDG derivative is conjugated to gold nanoparticles and anti-metadherin antibodies ([¹⁸F]-FDG-AuNP-Anti-MTDH) was described [170]. Radiolabelling was carried out in three steps but little information on the final purification of the nano-objects is given. [¹⁸F]-FDG-AuNP-Anti-MTDH were centrifuged, filtered (0.2 µm) before being dissolved in pure water. X-Ray analysis confirmed the presence of fluorine-18 on the [¹⁸F]-FDG-gold nanoparticles (FDG-AuNPs). The size and the ζ potential of the non-radioactive nanoparticles, [¹⁹F]-FDG-AuNP and [¹⁹F]-FDG-AuNP-Anti-MTDH, ranged from 15 to 40 nm and −13.30 to −18.06 mV, respectively, showing that antibody bioconjugation has no dramatic impact on the size and stability of the system.

### 4.2.4. Pd Nanosheets

An universal one-pot synthetic approach for the radiofluorination of biomolecules (HSA or nanobodies) as well as palladium nanosheets using [¹⁸F]-fluorothiophosphate (FTP) synths was very recently developed [125]. In the latter case, [¹⁸F]-FTP reacted with PEGylated Pd nanosheets (Pds-PEG) at room temperature for 15 min. The resulting mixture was purified by successive ultracentrifugations (13,000 rpm for 10 min, 3 times) until no radioactivity (due to unreacted [¹⁸F]-FTP and/or [¹⁸F]-F⁻) was detected in the supernatant. Radiochemical purity > 99% was monitored by radio-TLC analysis. No structural characterization of the [¹⁸F]-Pd nanosheets was given.

### 4.2.5. Silicon and Carbon-Based Nanoparticles

Different works on [¹⁸F]-labelled mesoporous silica nanoparticles (MSNPs) were also reported. Using the same synthesis and purification methodologies as those used for CeO₂ nanoparticles (vide supra), [¹⁸F]-MSNPs were prepared by grafting [¹⁸F]-SFB **38**) onto amino-functionalized MSNPs (30–130 nm) [143]. More recently, functionalized MSNPs (DBCO-PEG-MSNPs, DBCO = aza-dibenzocyclooctyne) were radiofluorinated using a pre-targeted strategy via a Cu free click conjugation (SPAAC reaction) [144]. The functionalized MSNPs were fully characterized. TEM showed that MSNPs were roughly spherical in shape, with an average diameter of 100–150 nm, and IR and ζ potential values confirmed
SiFA-maleimide-nanoparticles (green) and maleimide-nanoparticles (red) (analysis carried out with partially decayed [¹⁸F]-SiFA-maleimide-nanoparticles) (Reprinted with permission from Rapid ¹⁸F-labeling and loading of PEGylated gold nanoparticles for in vivo applications, Zhu, J.; Chin, J.; Wängler, C.; Wängler, B.; Lennox, R.B.; Schirrmacher, R., *Bioconjugate Chemistry* 2014, 25, 1143–1150, Copyright 2014, American Chemical Society).

Similar work in terms of purification and characterization was focused on the [¹⁸F]-radiolabelling of nitrone-gold nanoparticles using a bioorthogonal strain-promoted alkyne-nitrone cycloaddition (SPANC reaction) [124]. The radiofluorinated nanoparticles obtained from the SPANC reaction were passed through a PD-10 desalting column using 0.1M PBS buffer as eluent, leading to a pure [¹⁸F]-labelled gold nanoparticles solution ready to use. An extensive structural study (¹H NMR, IR, TEM and thermogravimetric analysis (TGA)) was carried out on non-radioactive nanoparticles (nitrone-gold nanoparticles and [¹⁹F]-labelled gold nanoparticles). Briefly, the nitrone-gold nanoparticles were spherical with a gold core of 2.6 ± 0.5 nm wide and perfectly monodisperse. Remarkably, the authors determined the raw formula of the nanoparticles (Au₅₀₀(MeO−EG₃−S⁻)₂₀₀(nitrone−EG₄−S⁻)₄₀, EG = ethylene glycol) as well as the amount of nitrone moieties per nanoparticle (0.840 µmol.mg⁻¹). The feasibility of the SPANC reaction was mainly demonstrated by NMR and XPS analyses on the isotopologous ¹⁹F model. The ¹⁹F NMR spectrum of [¹⁹F]-labelled gold nanoparticle recorded in D₂O showed only one peak at −222.91 ppm and comparison of the XPS spectra before and after conjugation exhibited the appearance of new peaks at 289.2 and 686.7 eV corresponding to C₁s and F₁s (C-F bond) for the [¹⁹F]-labelled gold nanoparticle, respectively. An average of 40 ¹⁹F atoms per nanoparticle were determined.

Finally, a less conventional nanoparticle architecture in which a [¹⁸F]-FDG derivative is conjugated to gold nanoparticles and anti-metadherin antibodies ([¹⁸F]-FDG-AuNP-Anti-MTDH) was described [170]. Radiolabelling was carried out in three steps but little information on the final purification of the nano-objects is given. [¹⁸F]-FDG-AuNP-Anti-MTDH were centrifuged, filtered (0.2 µm) before being dissolved in pure water. X-Ray analysis confirmed the presence of fluorine-18 on the [¹⁸F]-FDG-gold nanoparticles (FDG-AuNPs). The size and the *ζ* potential of the non-radioactive nanoparticles, [¹⁹F]-FDG-AuNP and [¹⁹F]-FDG-AuNP-Anti-MTDH, ranged from 15 to 40 nm and −13.30 to −18.06 mV, respectively, showing that antibody bioconjugation has no dramatic impact on the size and stability of the system.

### 4.2.4. Pd Nanosheets

An universal one-pot synthetic approach for the radiofluorination of biomolecules (HSA or nanobodies) as well as palladium nanosheets using [¹⁸F]-fluorothiophosphate (FTP) synths was very recently developed [125]. In the latter case, [¹⁸F]-FTP reacted with PEGylated Pd nanosheets (Pds-PEG) at room temperature for 15 min. The resulting mixture was purified by successive ultracentrifugations (13,000 rpm for 10 min, 3 times) until no radioactivity (due to unreacted [¹⁸F]-FTP and/or [¹⁸F]-F⁻) was detected in the supernatant. Radiochemical purity > 99% was monitored by radio-TLC analysis. No structural characterization of the [¹⁸F]-Pd nanosheets was given.

### 4.2.5. Silicon and Carbon-Based Nanoparticles

Different works on [¹⁸F]-labelled mesoporous silica nanoparticles (MSNPs) were also reported. Using the same synthesis and purification methodologies as those used for CeO₂ nanoparticles (vide supra), [¹⁸F]-MSNPs were prepared by grafting [¹⁸F]-SFB **38**) onto amino-functionalized MSNPs (30–130 nm) [143]. More recently, functionalized MSNPs (DBCO-PEG-MSNPs, DBCO = aza-dibenzocyclooctyne) were radiofluorinated using a pre-targeted strategy via a Cu free click conjugation (SPAAC reaction) [144]. The functionalized MSNPs were fully characterized. TEM showed that MSNPs were roughly spherical in shape, with an average diameter of 100–150 nm, and IR and *ζ* potential values confirmed
the functionalization of the silica nanoparticles (ζ-potential value of −7.63 mV for DBCO-PEG-MSNPs vs. +32.63 mV for $NH_2$-MSNPs). In this approach, as click conjugation (and therefore the radiolabelling of the nano-object) is carried out in vivo, the purification steps are limited to that of the radiofluorinated azide prosthetic group which was classically purified by reverse-phase HPLC.

[¹⁸F]-labelling of thermally hydrocarbonised porous silicon nanoparticles (THCPSi) is an interesting/unconventional example in that the nanoparticles are first radiolabelled before being coated with a fungal protein [137]. This two-step procedure required two (similar) purification processes. Thus, THCPSi were firstly incubated with [¹⁸F]-F⁻ for 10 min at 120 °C, followed by several cycles of centrifugation at 1500× g—wash in water/ethanol mixture using sonication, each lasting 20 min. Pure [¹⁸F]-THCPSi were collected in absolute ethanol, then coated with hydrophobin HFBII for 30 min at 80 °C. The final HFBII-[¹⁸F]-THCPSi particles were also purified by centrifugation followed by 3 washes in ultrapure water before being conditioned in sterile 0.9% NaCl. The coating obviously has an impact on the diameter of the [¹⁸F]-nanoparticles (average hydrodynamic size: 324 nm for HFBII-[¹⁸F]-THCPSi vs. 215 nm for [¹⁸F]-THCPSi), and on the ζ potential values, which remains increases slightly (from −33 mV to −21 mV). Still, the fungal protein coating of the HFBII-[¹⁸F]-THCPSi acted as a shield, resulting in a lower loss of [¹⁸F]-F⁻ ions in vivo (and therefore lower bone uptake) than for uncoated [¹⁸F]-THCPSi (0.04% for HFBII-[¹⁸F]-THCPSi vs. 0.21% for [¹⁸F]-THCPSi ID %/g at 60 min).

[¹⁸F]-nanodiamonds were studied by using radiolabelling and purification methods identical to those previously reported by the same group for ceria [89] or mesoporous silica nanoparticles [143] (purification by successive centrifugation/washing cycles until no radioactivity was detected in the supernatant) [130]. Remarkably, the authors proposed (in the supplementary data of [130]) a structural study of these nano-objects with fluorine-19. [¹⁹F]-nanodiamonds were prepared and purified in the same way as [¹⁸F]-nanodiamonds, then analysed in detail by AFM, TEM, IR and ¹⁹F NMR. Using an NMR internal standard [¹⁹F]-NaF, a percentage of 4.3% of fluorine-labelled groups in [¹⁹F]-labelled nanodiamond was assessed (NMR study carried out in deuterated methanol).

Radiofluorinated PEG-functionalized nano-graphene oxides ([¹⁸F]-nGO-PEG) were synthetized and studied as PET probes [131]. nGO-PEG were easily radiolabelled by being incubated with [¹⁸F]-F⁻ for 1 h at 80 °C in acidic conditions. Unreacted [¹⁸F]-F⁻ was classically removed by filtration using 0.2 µm filter and centrifugation/washes in saline solution cycles until no radioactivity was detect in the filtrate. The efficiency of the radiolabelling was monitored using radio-TLC (eluent: EtOAc/n-hexane; no migration for [¹⁸F]-nGO-PEG, R_f = 90 mm for [¹⁸F]-F⁻). Non-fluorinated nGOs were characterized by XRD profiles, ζ potential values and TEM images. More interestingly, the efficiency of radiolabelling was validated on [¹⁹F]-nGO-PEG by XPS (F₁s peak at approximately 725 eV) and also by IR. Indeed, the presence of fluorine is confirmed by (i) a sharp decrease in the intensity of the band at 3429 cm⁻¹ due to the disappearance of hydroxyl groups (substituted by fluorine) and (ii) in the 1100–1150 cm⁻¹ region, the disappearance of the C-O band and the appearance of the C-F band.

These last two examples perfectly illustrate the importance of conducting in-depth structural studies on non-radioactive nanoparticles ([¹⁹F]-labelled) in parallel with the radiofluorination investigation.

In conclusion, due to the relatively short lifetime of fluorine-18, the purification step, like the radiolabelling method, needs to be simple, efficient, and as fast as possible to obtain a [¹⁸F]-radiolabelled compound of high radiochemical purity. On the basis of the examples cited above, it seems unarguable that whatever the intrinsic nature of the nanoparticle, the main purification criterion taken into account is its size/molecular weight, making steric exclusion chromatography or (ultra)centrifugation the two gold-standard methods for purifying radiolabelled nano-objects. However, for magnetic nanoparticles, magnetic separation with a strong NdFeB magnet can be an advantageous alternative to
the functionalization of the silica nanoparticles (ζ-potential value of −7.63 mV for DBCO-PEG-MSNPs vs. +32.63 mV for $NH_2$-MSNPs). In this approach, as click conjugation (and therefore the radiolabelling of the nano-object) is carried out in vivo, the purification steps are limited to that of the radiofluorinated azide prosthetic group which was classically purified by reverse-phase HPLC.

[¹⁸F]-labelling of thermally hydrocarbonised porous silicon nanoparticles (THCPSi) is an interesting/unconventional example in that the nanoparticles are first radiolabelled before being coated with a fungal protein [137]. This two-step procedure required two (similar) purification processes. Thus, THCPSi were firstly incubated with [¹⁸F]-F⁻ for 10 min at 120 °C, followed by several cycles of centrifugation at 1500× g—wash in water/ethanol mixture using sonication, each lasting 20 min. Pure [¹⁸F]-THCPSi were collected in absolute ethanol, then coated with hydrophobin HFBII for 30 min at 80 °C. The final HFBII-[¹⁸F]-THCPSi particles were also purified by centrifugation followed by 3 washes in ultrapure water before being conditioned in sterile 0.9% NaCl. The coating obviously has an impact on the diameter of the [¹⁸F]-nanoparticles (average hydrodynamic size: 324 nm for HFBII-[¹⁸F]-THCPSi vs. 215 nm for [¹⁸F]-THCPSi), and on the ζ potential values, which remains increases slightly (from −33 mV to −21 mV). Still, the fungal protein coating of the HFBII-[¹⁸F]-THCPSi acted as a shield, resulting in a lower loss of [¹⁸F]-F⁻ ions in vivo (and therefore lower bone uptake) than for uncoated [¹⁸F]-THCPSi (0.04% for HFBII-[¹⁸F]-THCPSi vs. 0.21% for [¹⁸F]-THCPSi ID %/g at 60 min).

[¹⁸F]-nanodiamonds were studied by using radiolabelling and purification methods identical to those previously reported by the same group for ceria [89] or mesoporous silica nanoparticles [143] (purification by successive centrifugation/washing cycles until no radioactivity was detected in the supernatant) [130]. Remarkably, the authors proposed (in the supplementary data of [130]) a structural study of these nano-objects with fluorine-19. [¹⁹F]-nanodiamonds were prepared and purified in the same way as [¹⁸F]-nanodiamonds, then analysed in detail by AFM, TEM, IR and ¹⁹F NMR. Using an NMR internal standard [¹⁹F]-NaF, a percentage of 4.3% of fluorine-labelled groups in [¹⁹F]-labelled nanodiamond was assessed (NMR study carried out in deuterated methanol).

Radiofluorinated PEG-functionalized nano-graphene oxides ([¹⁸F]-nGO-PEG) were synthetized and studied as PET probes [131]. nGO-PEG were easily radiolabelled by being incubated with [¹⁸F]-F⁻ for 1 h at 80 °C in acidic conditions. Unreacted [¹⁸F]-F⁻ was classically removed by filtration using 0.2 µm filter and centrifugation/washes in saline solution cycles until no radioactivity was detect in the filtrate. The efficiency of the radiolabelling was monitored using radio-TLC (eluent: EtOAc/n-hexane; no migration for [¹⁸F]-nGO-PEG, R_f = 90 mm for [¹⁸F]-F⁻). Non-fluorinated nGOs were characterized by XRD profiles, ζ potential values and TEM images. More interestingly, the efficiency of radiolabelling was validated on [¹⁹F]-nGO-PEG by XPS (F₁s peak at approximately 725 eV) and also by IR. Indeed, the presence of fluorine is confirmed by (i) a sharp decrease in the intensity of the band at 3429 cm⁻¹ due to the disappearance of hydroxyl groups (substituted by fluorine) and (ii) in the 1100–1150 cm⁻¹ region, the disappearance of the C-O band and the appearance of the C-F band.

These last two examples perfectly illustrate the importance of conducting in-depth structural studies on non-radioactive nanoparticles ([¹⁹F]-labelled) in parallel with the radiofluorination investigation.

In conclusion, due to the relatively short lifetime of fluorine-18, the purification step, like the radiolabelling method, needs to be simple, efficient, and as fast as possible to obtain a [¹⁸F]-radiolabelled compound of high radiochemical purity. On the basis of the examples cited above, it seems unarguable that whatever the intrinsic nature of the nanoparticle, the main purification criterion taken into account is its size/molecular weight, making steric exclusion chromatography or (ultra)centrifugation the two gold-standard methods for purifying radiolabelled nano-objects. However, for magnetic nanoparticles, magnetic separation with a strong NdFeB magnet can be an advantageous alternative to
steric exclusion chromatography or centrifugation (in which aggregation of nanoparticles
can occur).

In addition, as mentioned in a recent and very comprehensive review on nanoparticles
characterization techniques in radiopharmaceutical sciences [164], the following statement
shall be strongly considered. "In general, there is a drastic lack of extensive character-
ization of nanoparticles used in radiopharmaceuticals sciences. In some works, these
characterizations have not even been indicated". While it is true that in many studies, the
structural characterization of nanoparticles was more or less complete, or even minimalist,
and very often focused on the nanoparticle before radiolabelling, more substantial studies
(carried out on [¹⁸F]-labelled and/or cold [¹⁹F]-labelled nanoparticles) highlighted the
(radio)fluorination (by XPS study in particular) and/or the number of fluorine atoms/ions
coupled to the nanoparticle. On the other hand, the integrity of the nanoparticles structure
is often little discussed/controlled, whereas the addition of fluorine-18 or a radiofluori-
nated prosthetic group can have a real impact on the structure of the nanoparticle itself,
as does the radiofluorination method used. This criterion therefore remains to be better
understood in the design of future radiofluorinated nano-objects. Finally, it seems essential
to characterize all the new radiolabelled nanoparticles as precisely as possible because the
better the structural characterization of the object is, (i) the easier it will be to understand
the structure/in vivo uptake relationship, (ii) the more controlled the radiolabelling stage
will be (minimum quantity of radioactivity to be introduced into the nanoparticle, rapid
purification, etc.), and (iii) the faster the translation from the bench to the bedside can
be achieved.

**5. Discussion**

As illustrated above, a large number of publications concern the radiofluorination
of nanopatterns, spanning all types of nanoparticles from organic nanopatterns such
as micelles, emulsions, liposomes, and polymers, to inorganic nanoparticles. The latter
are either inert, such as silica, or display intrinsic physical properties such as some carbon
or silicon-based nanopatterns, gold, iron oxide and rare earth nanoparticles, or even
quantum dots. Their sizes and shapes are diverse as well as their surface coatings, hence
surface charges. Given the non-invasive character of radio imaging, the radiolabelling
of these nanopatterns was used for monitoring them in vivo and understanding their
behaviour or predicting their therapeutic effectiveness. Nanoparticle radiofluorination was
also envisaged to develop contrast agents for in vivo and in vitro imaging, as nanoparticles
offer significant advantages over small molecules, such as the generation of a stronger
and longer-lasting contrast. Different approaches have been developed aiming at efficient
labelling and purification methods to reduce the overall time to the target, which is key in
radio imaging.

As illustrated in the Section 3 of this manuscript, the main approaches to radiofluori-
nated NPs are:

* direct radiofluorination either by bombardment of [¹⁸O] containing nanoparticles, reaction of [¹⁸F]-fluoride with the inorganic core of the nanoparticles or incorporation of radiolabelled constituents during their synthesis (see Section 3.2) and,

* introduction of an organic based radiofluorinated compound by supramolecular interaction or covalent bonding, this last conjugation being achieved either classically in vitro or more recently using in vivo pre-targeting approaches (see Section 3.3).

Each approach obviously presents its pros and cons. For example, methods involving
the direct generation of the nanoparticles with radiolabelled materials (as described in
Section 3.2.3) are synthetically classical from the point of view of nanoparticles generation.
Yet, due to the short half-life of [¹⁸F]-fluorine, they mainly exhibit moderate radiochemical
yields (especially not decay corrected). Still, they do represent interesting approaches to
generate new tools and evaluate their ability to perform the intended imaging. However,
concerns may appear for routine preclinical, and even more for clinical, investigations. This
is due not only to the production process leading to low radiochemical yields but also to
steric exclusion chromatography or centrifugation (in which aggregation of nanoparticles
can occur).

In addition, as mentioned in a recent and very comprehensive review on nanoparticles
characterization techniques in radiopharmaceutical sciences [164], the following statement
shall be strongly considered. "In general, there is a drastic lack of extensive character-
ization of nanoparticles used in radiopharmaceuticals sciences. In some works, these
characterizations have not even been indicated". While it is true that in many studies, the
structural characterization of nanoparticles was more or less complete, or even minimalist,
and very often focused on the nanoparticle before radiolabelling, more substantial studies
(carried out on [¹⁸F]-labelled and/or cold [¹⁹F]-labelled nanoparticles) highlighted the
(radio)fluorination (by XPS study in particular) and/or the number of fluorine atoms/ions
coupled to the nanoparticle. On the other hand, the integrity of the nanoparticles structure
is often little discussed/controlled, whereas the addition of fluorine-18 or a radiofluori-
nated prosthetic group can have a real impact on the structure of the nanoparticle itself,
as does the radiofluorination method used. This criterion therefore remains to be better
understood in the design of future radiofluorinated nano-objects. Finally, it seems essential
to characterize all the new radiolabelled nanoparticles as precisely as possible because the
better the structural characterization of the object is, (i) the easier it will be to understand
the structure/in vivo uptake relationship, (ii) the more controlled the radiolabelling stage
will be (minimum quantity of radioactivity to be introduced into the nanoparticle, rapid
purification, etc.), and (iii) the faster the translation from the bench to the bedside can
be achieved.

**5. Discussion**

As illustrated above, a large number of publications concern the radiofluorination
of nanoplatforms, spanning all types of nanoparticles from organic nanoplatforms such
as micelles, emulsions, liposomes, and polymers, to inorganic nanoparticles. The latter
are either inert, such as silica, or display intrinsic physical properties such as some carbon
or silicon-based nanoplatforms, gold, iron oxide and rare earth nanoparticles, or even
quantum dots. Their sizes and shapes are diverse as well as their surface coatings, hence
surface charges. Given the non-invasive character of radio imaging, the radiolabelling
of these nanoplatforms was used for monitoring them in vivo and understanding their
behaviour or predicting their therapeutic effectiveness. Nanoparticle radiofluorination was
also envisaged to develop contrast agents for in vivo and in vitro imaging, as nanoparticles
offer significant advantages over small molecules, such as the generation of a stronger
and longer-lasting contrast. Different approaches have been developed aiming at efficient
labelling and purification methods to reduce the overall time to the target, which is key in
radio imaging.

As illustrated in the Section 3 of this manuscript, the main approaches to radiofluori-
nated NPs are:

* direct radiofluorination either by bombardment of [¹⁸O] containing nanoparticles, reaction of [¹⁸F]-fluoride with the inorganic core of the nanoparticles or incorporation of radiolabelled constituents during their synthesis (see Section 3.2) and,

* introduction of an organic based radiofluorinated compound by supramolecular interaction or covalent bonding, this last conjugation being achieved either classically in vitro or more recently using in vivo pre-targeting approaches (see Section 3.3).

Each approach obviously presents its pros and cons. For example, methods involving
the direct generation of the nanoparticles with radiolabelled materials (as described in
Section 3.2.3) are synthetically classical from the point of view of nanoparticles generation.
Yet, due to the short half-life of [¹⁸F]-fluorine, they mainly exhibit moderate radiochemical
yields (especially not decay corrected). Still, they do represent interesting approaches to
generate new tools and evaluate their ability to perform the intended imaging. However,
concerns may appear for routine preclinical, and even more for clinical, investigations. This
is due not only to the production process leading to low radiochemical yields but also to
the difficulty to perform extensive analyses to assess the quality of the radiofluorinated probes, due to obvious radioprotection issues.

From that perspective, radiofluorination of pre-formed and well-characterized nanoplat-
forms, can appear more promising. Indeed, their absence of toxicity can be assessed prior
to radiolabelling (even though a different behaviour of radiolabelled nanoparticle formula-
tions compared to their unmodified analogues cannot be ruled out).

An interesting case of nanoparticles [¹⁸F]-radiolabelling is illustrated by the direct bombardment of inorganic nanoparticles incorporating ¹⁸O atoms. This method has been described to proceed with a good efficiency. Its main issue remains the generation of a range of radioactive elements (other than ¹⁸F, such as ¹³N, ⁴⁴gSc, etc.) depending on the architecture of the nanoparticle. As the elements described are short lived positron emitters, the impact on dosimetry should be limited, but imaging might be affected by the different decays making the quantitative analysis less straightforward.

Late-stage introduction of a radiolabelled motif represents a common alternative. Different routes have been explored. Direct introduction of [¹⁸F]-fluoride appears especially valuable for inorganic based nanoparticles as it represents a short path towards radiolabelled nanoparticles (Section 3.2.2). It is of obvious interest in terms of production time, especially when the reaction can be carried out under aqueous conditions. Still the kinetics of the radiolabelling can be highly dependent on the nature of the nanoparticle and its coating. Moreover, the impact of the radiofluorination conditions on the physico-chemical characteristics of the nanoparticles, even if low molar amounts of fluoride are engaged, needs to be established in a repeated number of experiments (to obtain meaningful statistics).

An alternative method consists in the preparation of a radiofluorinated prosthetic
group, that is further coupled to a properly surface engineered nanoparticle to display
reactive functional groups (Section 3.3.2). This approach parallels the radiolabelling of
biomacromolecules such as peptides or proteins. It is an obvious valuable strategy when
a highly efficient synthesis of the radiofluorinated prosthetic group is available from
the production site. It is also unavoidable when the common radiofluorination by C–F
bond formation (namely significant heating in polar solvents and basic conditions) can be
deleterious for the nanoparticles (as it is for biomacromolecules).

Lastly, another essential factor associated with [¹⁸F]-fluorine labelling for in vivo
imaging is that the time constraint related to the half-life time of this isotope shall not
be exclusively seen from the point of view of the preparation and purification of the
radiolabelled material. It is also important to consider the time devoted to its analysis
prior to in vivo injection, and most importantly for large species, the time of biodistribution
to the desired target and clearance from the blood stream. These considerations seem
to have restrained the development of nanoparticles in [¹⁸F]-PET imaging. Indeed, ex
vivo prepared radiofluorinated nanoparticles described in Sections 3.3.1 and 3.3.2 have
an imaging efficiency that strongly depends on their time to target. Nevertheless, an
upcoming development shall now arise from the latest trends of in vivo labelling relying
on bio-orthogonal chemical ligation (see Section 3.3.2). These methods are appearing as
highly appropriate approaches. Indeed, they consist in a first injection of a pre-targeted
nanoparticle, allowing the biodistribution of a perfectly characterized material. This is
then followed (after the time required for the nanoparticle to reach the biological target)
by the injection of a small molecule carrying the radioactive fluorine and the functional
group complementary to one carried by the pre-targeted nanoparticle. It is noteworthy that
contrarily to nanoprobes, very efficient analysis and purification techniques are available
for molecular probes. Thus, this approach combines the best of both worlds in terms of
synthesis, characterization and biodistribution.

However, two main limitations remain. The bio-orthogonal conjugation needs to
present an extremely fast kinetic profile, and the accumulation of the radiolabelled probes
needs to be driven only by the pre-targeting functions, and not by non-specific interac-
tions. Advantageously, these latter issues have been circumvented in the related topic of
the difficulty to perform extensive analyses to assess the quality of the radiofluorinated probes, due to obvious radioprotection issues.

From that perspective, radiofluorination of pre-formed and well-characterized nanoplat-
forms, can appear more promising. Indeed, their absence of toxicity can be assessed prior
to radiolabelling (even though a different behaviour of radiolabelled nanoparticle formula-
tions compared to their unmodified analogues cannot be ruled out).

An interesting case of nanoparticles [¹⁸F]-radiolabelling is illustrated by the direct bombardment of inorganic nanoparticles incorporating ¹⁸O atoms. This method has been described to proceed with a good efficiency. Its main issue remains the generation of a range of radioactive elements (other than ¹⁸F, such as ¹³N, ⁴⁴gSc, etc.) depending on the architecture of the nanoparticle. As the elements described are short lived positron emitters, the impact on dosimetry should be limited, but imaging might be affected by the different decays making the quantitative analysis less straightforward.

Late-stage introduction of a radiolabelled motif represents a common alternative. Different routes have been explored. Direct introduction of [¹⁸F]-fluoride appears especially valuable for inorganic based nanoparticles as it represents a short path towards radiolabelled nanoparticles (Section 3.2.2). It is of obvious interest in terms of production time, especially when the reaction can be carried out under aqueous conditions. Still the kinetics of the radiolabelling can be highly dependent on the nature of the nanoparticle and its coating. Moreover, the impact of the radiofluorination conditions on the physico-chemical characteristics of the nanoparticles, even if low molar amounts of fluoride are engaged, needs to be established in a repeated number of experiments (to obtain meaningful statistics).

An alternative method consists in the preparation of a radiofluorinated prosthetic
group, that is further coupled to a properly surface engineered nanoparticle to display
reactive functional groups (Section 3.3.2). This approach parallels the radiolabelling of
biomacromolecules such as peptides or proteins. It is an obvious valuable strategy when
a highly efficient synthesis of the radiofluorinated prosthetic group is available from
the production site. It is also unavoidable when the common radiofluorination by C–F
bond formation (namely significant heating in polar solvents and basic conditions) can be
deleterious for the nanoparticles (as it is for biomacromolecules).

Lastly, another essential factor associated with [¹⁸F]-fluorine labelling for in vivo
imaging is that the time constraint related to the half-life time of this isotope shall not
be exclusively seen from the point of view of the preparation and purification of the
radiolabelled material. It is also important to consider the time devoted to its analysis
prior to in vivo injection, and most importantly for large species, the time of biodistribution
to the desired target and clearance from the blood stream. These considerations seem
to have restrained the development of nanoparticles in [¹⁸F]-PET imaging. Indeed, ex
vivo prepared radiofluorinated nanoparticles described in Sections 3.3.1 and 3.3.2 have
an imaging efficiency that strongly depends on their time to target. Nevertheless, an
upcoming development shall now arise from the latest trends of in vivo labelling relying
on bio-orthogonal chemical ligation (see Section 3.3.2). These methods are appearing as
highly appropriate approaches. Indeed, they consist in a first injection of a pre-targeted
nanoparticle, allowing the biodistribution of a perfectly characterized material. This is
then followed (after the time required for the nanoparticle to reach the biological target)
by the injection of a small molecule carrying the radioactive fluorine and the functional
group complementary to one carried by the pre-targeted nanoparticle. It is noteworthy that
contrarily to nanoprobes, very efficient analysis and purification techniques are available
for molecular probes. Thus, this approach combines the best of both worlds in terms of
synthesis, characterization and biodistribution.

However, two main limitations remain. The bio-orthogonal conjugation needs to
present an extremely fast kinetic profile, and the accumulation of the radiolabelled probes
needs to be driven only by the pre-targeting functions, and not by non-specific interac-
tions. Advantageously, these latter issues have been circumvented in the related topic of
pre-targeted antibodies for PET imaging which will benefit to further developments for
nanoparticles radiofluorination [171].

Similar to the radiolabelling step, the purification of radiofluorinated nanoparticles must be rapid and efficient, and the process must be transposable to automated radiosynthesis. Most [¹⁸F]-nanoparticles are purified using size-exclusion chromatography or (ultra)centrifugation. Although a few elegant alternatives have been identified (direct proton beam irradiation requiring no purification except waiting for the decay of radioactive by-products produced during the proton irradiation, or magnetic separation), they are unfortunately very nanoparticle-dependent and cannot be transposed to the vast majority of those.

The characterization of fluorine-18 radiolabelled nanoparticles is undoubtedly the step
that needs to be optimised/worked on for a future clinical routine usage, in order to meet
the requirements of international authorities such as the European Medicines Agency or the
Food and Drug Administration. To optimize radioprotection, and because a large number
of analytical techniques cannot be performed on radioactive materials, the optimization of
this stage necessarily involves a detailed structural study of [¹⁹F]-analogues using a set of
numerous analytical techniques, as well as structural comparisons between [¹⁹F]-labelled
and non-labelled systems (supposing first that the nanoparticle to be labelled has been
carefully characterized, and that its stability in biological media, such as cell culture or
plasma, has been ascertained). Care should be taken to generate the [¹⁹F]-analogue in as
identical conditions as possible to those of [¹⁸F]-radiolabelling. This raises technical issues
to bridge the low concentrations used in radiolabelling processes and the low sensitivity
of the characterization techniques needed to ascertain the physico-chemical integrity of
the nanoparticles, especially inorganic ones. Ensuring that data are transposable is thus a
huge challenge. The methodology used for the structural characterization of generation
4 PAMAM dendrimers radiolabelled with fluorine-18 [55,56] is certainly an example to
follow. However, this example surely represents the ideal case, given that a dendrimer is a
macromolecular system which is easier to characterize than other types of nanoparticles.

Once purity assessed, lack of stability is the next potential bottle-neck. Indeed, release
of the radiolabel in vivo would alter the biodistribution of the radioactivity which would no
longer reflect the sole fate of the radiolabelled nanoparticles. Nuclear imaging's relevance
to nanotherapeutic translation thus depends on developments in radiolabelling strategies
to obtain a high radiolabelling stability. First evaluations in vitro could point out a potential
lack of stability. It could arise from the release of [¹⁸F]-fluoride, prosthetic group, or of
the nanoparticle coating used to graft this prosthetic group, or of the fluorinated tracer in
case of labelling via encapsulation. Such studies are scarce. For example, the [¹⁸F]-fluoride
loading stability of superparamagnetic iron oxide nanoparticles, coated with polyacrylic
acid and aluminium hydroxide, was tested in vitro in longitudinal experiments up to 4 h
using water, buffer, and cell culture media at 37 °C [82]. After efficient synthesis of novel
[¹⁸F]-labelled imaging agents based on YF₃ nanoparticles, [¹⁸F]-labelled YF₃ nanoparticles
stability was studied in mouse and human serum [62]. In addition to study stability in
buffer or storage medium, studies in biological medium as cell culture media, or best in
plasma, are necessary because of the large number of interfering molecules in blood. These
studies must be performed at 37 °C and over a period in accordance with future in vivo
studies to be predictive of what will happen in vivo. This type of studies is particularly
necessary for labelling strategies that rely on non-covalent binding of ¹⁸F.

Importantly in vitro studies need to be supplemented by cellular and in vivo studies
to evaluate metabolism (e.g., hepatic) that could lead to radionuclide release. It could thus
be interesting to perform blood and post-mortem tissue analysis to evaluate radiolabelling
stability. On the reverse, as free fluoride is efficiently taken up in bones, quantitative in vivo
PET imaging allows an indirect evaluation of the radiolabelling stability (but only as far as
[¹⁸F]-fluoride release is concerned). As well the integrity of the nanoparticle needs to be
ascertained. Some studies indeed report the detection of degradation products [16,89].
pre-targeted antibodies for PET imaging which will benefit to further developments for
nanoparticles radiofluorination [171].

Similar to the radiolabelling step, the purification of radiofluorinated nanoparticles must be rapid and efficient, and the process must be transposable to automated radiosynthesis. Most [¹⁸F]-nanoparticles are purified using size-exclusion chromatography or (ultra)centrifugation. Although a few elegant alternatives have been identified (direct proton beam irradiation requiring no purification except waiting for the decay of radioactive by-products produced during the proton irradiation, or magnetic separation), they are unfortunately very nanoparticle-dependent and cannot be transposed to the vast majority of those.

The characterization of fluorine-18 radiolabelled nanoparticles is undoubtedly the step
that needs to be optimised/worked on for a future clinical routine usage, in order to meet
the requirements of international authorities such as the European Medicines Agency or the
Food and Drug Administration. To optimize radioprotection, and because a large number
of analytical techniques cannot be performed on radioactive materials, the optimization of
this stage necessarily involves a detailed structural study of [¹⁹F]-analogues using a set of
numerous analytical techniques, as well as structural comparisons between [¹⁹F]-labelled
and non-labelled systems (supposing first that the nanoparticle to be labelled has been
carefully characterized, and that its stability in biological media, such as cell culture or
plasma, has been ascertained). Care should be taken to generate the [¹⁹F]-analogue in as
identical conditions as possible to those of [¹⁸F]-radiolabelling. This raises technical issues
to bridge the low concentrations used in radiolabelling processes and the low sensitivity
of the characterization techniques needed to ascertain the physico-chemical integrity of
the nanoparticles, especially inorganic ones. Ensuring that data are transposable is thus a
huge challenge. The methodology used for the structural characterization of generation
4 PAMAM dendrimers radiolabelled with fluorine-18 [55,56] is certainly an example to
follow. However, this example surely represents the ideal case, given that a dendrimer is a
macromolecular system which is easier to characterize than other types of nanoparticles.

Once purity assessed, lack of stability is the next potential bottle-neck. Indeed, release
of the radiolabel in vivo would alter the biodistribution of the radioactivity which would no
longer reflect the sole fate of the radiolabelled nanoparticles. Nuclear imaging's relevance
to nanotherapeutic translation thus depends on developments in radiolabelling strategies
to obtain a high radiolabelling stability. First evaluations in vitro could point out a potential
lack of stability. It could arise from the release of [¹⁸F]-fluoride, prosthetic group, or of
the nanoparticle coating used to graft this prosthetic group, or of the fluorinated tracer in
case of labelling via encapsulation. Such studies are scarce. For example, the [¹⁸F]-fluoride
loading stability of superparamagnetic iron oxide nanoparticles, coated with polyacrylic
acid and aluminium hydroxide, was tested in vitro in longitudinal experiments up to 4 h
using water, buffer, and cell culture media at 37 °C [82]. After efficient synthesis of novel
[¹⁸F]-labelled imaging agents based on YF₃ nanoparticles, [¹⁸F]-labelled YF₃ nanoparticles
stability was studied in mouse and human serum [62]. In addition to study stability in
buffer or storage medium, studies in biological medium as cell culture media, or best in
plasma, are necessary because of the large number of interfering molecules in blood. These
studies must be performed at 37 °C and over a period in accordance with future in vivo
studies to be predictive of what will happen in vivo. This type of studies is particularly
necessary for labelling strategies that rely on non-covalent binding of ¹⁸F.

Importantly in vitro studies need to be supplemented by cellular and in vivo studies
to evaluate metabolism (e.g., hepatic) that could lead to radionuclide release. It could thus
be interesting to perform blood and post-mortem tissue analysis to evaluate radiolabelling
stability. On the reverse, as free fluoride is efficiently taken up in bones, quantitative in vivo
PET imaging allows an indirect evaluation of the radiolabelling stability (but only as far as
[¹⁸F]-fluoride release is concerned). As well the integrity of the nanoparticle needs to be
ascertained. Some studies indeed report the detection of degradation products [16,89].
Then, even if the doses injected are very low (some mCi representing few pmol), the toxicity of the radiolabelled nanoplatform should be assessed. It is generally admitted that radiolabelling is unlikely to alter toxicity, and that it is rather the toxicity of the nanoparticle that must be evaluated. This can be done easily on the non-labelled nanoparticles (in case of labelling strategies that involve pre-formed nanoplatforms). Cell survival after exposure to different concentrations of nanoparticles and cell proliferation at different days post-exposition to nanoparticles could be studied as done by González-Gómez et al. to study superparamagnetic iron oxide nanoparticles [82]. However, one should keep in mind that the radiolabelling step may change the physico-chemical characteristics of the nanoplatform such as hydrodynamic size, surface charge, hydrophilicity balance...all parameters that may alter their interaction with cells and change their metabolism. Study of the toxicity of the [¹⁹F]-analogue, prepared under identical conditions, would be the best approach.

**6. Conclusions and Future Directions**

Our study of the literature shows a renewed interest for the radiofluorination of
nanoplatforms in the past decade, flowering out of a continuously evolving radiofluorina-
tion ground (especially in the field of user-friendly radiofluorination of small molecules
leveraging the labelling of nanoplatforms). These technologies are now mature to translate
to highly sophisticated health-related uses. It appears that based on these evolutions,
radiofluorinated nanoplatforms are especially suited when their local injection is envisaged,
as reported in a recent work [172]. The interest in radiofluorination of nanoplatforms is
also prompted by the development of pre-targeting approaches. This two-step technology
consists first in the administration of the nanoplatform. Once the nanoplatform has dis-
seminated in the body, and accumulated in the region of interest, a molecular radiotracer is
administrated. The elegance of the process lies in the fact that both the nanoplatform and
the molecular radiotracer are equipped with functional groups that will selectively react
with each other, leading to their specific recognition in vitro and in vivo. Radioactivity thus
accumulates in the region of interest, which can be imaged with high accuracy.

Provided that optimal in vivo bioconjugation conditions are found (proportion of
the two components, time interval between the two injections, stable bioconjugation,
neglectable non-specific interactions, etc.), the advantages should be numerous: (i) a range
of effective and rapid bioorthogonal conjugation methods is available, (ii) characterization
is simplified to purification of the radiofluorinated prosthetic group (using well-mastered
chromatographic techniques) and that of the modified nanoparticle, and finally (iii) the
technology can be applied to a large number of nanoparticles and is compatible with the
short-lived radionuclides and relatively slow pharmacokinetics of nanoparticles (maximum
tumour uptake after a few hours/days).

The implementation of this technology has emerged owing to the tremendous progress
made in the surface engineering of nanoparticles, and taking advantage of the methodolo-
gies developed for macromolecular systems [173,174]. If properly performed, it should
allow access to a wide range of diagnostic (bi- and multimodal imaging) or theranostic
combinations, especially in the case of intrinsically active nanoparticles leading to improved
monitoring of treatments. Based on imaging results, the treatment strategy can be adapted,
with a direct impact on patient management, by protecting patients from unnecessary side
effects and costs.

Thus, radio imaging of nanoparticles can play an important role in the management of
the individual patient and novel radiofluorination methods can help to achieve this goal.

**Author Contributions:** Project conceptualization and administration: C.A.; data collection and investigation: C.A., Ē.B., M.D. and E.G.; manuscript drafting: C.A., Ē.B., M.D., E.G. and I.Q.; highlight drafting: M.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** We thank CNRS: INSERM and UPS for their financial support. M. Deleuzière thanks the University of Toulouse and Occitanie Region for her Ph.D. funding (n°00138007). This work has been
Then, even if the doses injected are very low (some mCi representing few pmol), the toxicity of the radiolabelled nanoplatform should be assessed. It is generally admitted that radiolabelling is unlikely to alter toxicity, and that it is rather the toxicity of the nanoparticle that must be evaluated. This can be done easily on the non-labelled nanoparticles (in case of labelling strategies that involve pre-formed nanoplatforms). Cell survival after exposure to different concentrations of nanoparticles and cell proliferation at different days post-exposition to nanoparticles could be studied as done by González-Gómez et al. to study superparamagnetic iron oxide nanoparticles [82]. However, one should keep in mind that the radiolabelling step may change the physico-chemical characteristics of the nanoplatform such as hydrodynamic size, surface charge, hydrophilicity balance...all parameters that may alter their interaction with cells and change their metabolism. Study of the toxicity of the [¹⁹F]-analogue, prepared under identical conditions, would be the best approach.

**6. Conclusions and Future Directions**

Our study of the literature shows a renewed interest for the radiofluorination of nanoplatforms in the past decade, flowering out of a continuously evolving radiofluorination ground (especially in the field of user-friendly radiofluorination of small molecules leveraging the labelling of nanoplatforms). These technologies are now mature to translate to highly sophisticated health-related uses. It appears that based on these evolutions, radiofluorinated nanoplatforms are especially suited when their local injection is envisaged, as reported in a recent work [172]. The interest in radiofluorination of nanoplatforms is also prompted by the development of pre-targeting approaches. This two-step technology consists first in the administration of the nanoplatform. Once the nanoplatform has disseminated in the body, and accumulated in the region of interest, a molecular radiotracer is administrated. The elegance of the process lies in the fact that both the nanoplatform and the molecular radiotracer are equipped with functional groups that will selectively react with each other, leading to their specific recognition in vitro and in vivo. Radioactivity thus accumulates in the region of interest, which can be imaged with high accuracy.

Provided that optimal in vivo bioconjugation conditions are found (proportion of the two components, time interval between the two injections, stable bioconjugation, neglectable non-specific interactions, etc.), the advantages should be numerous: (i) a range of effective and rapid bioorthogonal conjugation methods is available, (ii) characterization is simplified to purification of the radiofluorinated prosthetic group (using well-mastered chromatographic techniques) and that of the modified nanoparticle, and finally (iii) the technology can be applied to a large number of nanoparticles and is compatible with the short-lived radionuclides and relatively slow pharmacokinetics of nanoparticles (maximum tumour uptake after a few hours/days).

The implementation of this technology has emerged owing to the tremendous progress made in the surface engineering of nanoparticles, and taking advantage of the methodologies developed for macromolecular systems [173,174]. If properly performed, it should allow access to a wide range of diagnostic (bi- and multimodal imaging) or theranostic combinations, especially in the case of intrinsically active nanoparticles leading to improved monitoring of treatments. Based on imaging results, the treatment strategy can be adapted, with a direct impact on patient management, by protecting patients from unnecessary side effects and costs.

Thus, radio imaging of nanoparticles can play an important role in the management of the individual patient and novel radiofluorination methods can help to achieve this goal.

**Author Contributions:** Project conceptualization and administration: C.A.; data collection and investigation: C.A., É.B., M.D. and E.G.; manuscript drafting: C.A., É.B., M.D., E.G. and I.Q.; highlight drafting: M.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** We thank CNRS: INSERM and UPS for their financial support. M. Deleuzière thanks the University of Toulouse and Occitanie Region for her Ph.D. funding (n°00138007). This work has been
labelled through the grant EUR CARe N°ANR-18-EURE-0003 in the framework of the Programme des Investissements d'Avenir.

**Conflicts of Interest:** The authors declare no conflicts of interest.

**Nomenclature**

**Radiochemical Purity (RCP):** the proportion of the total radioactivity in the sample that is present as the desired radiolabelled species (definition from the International Atomic Energy Agency).

**Molar radioactivity:** the ratio of the amount of radiofluorinated, [¹⁸F]-containing, to the amount of fluorinated, ¹⁹F and ¹⁸F containing, compound.

**Specific radioactivity:** the activity per unit mass of a radionuclide is a physical property of the specified radionuclide and is given by the equation: $a[Bq/g] = \frac{N_A \ln2}{M_{t1/2}}$, where N<sub>A</sub> is the Avogadro constant, M is the molar mass of the nuclide, and t<sub>1/2</sub> the half-life time of the radionuclide. For ¹⁸F, a = 1.71 × 10⁹ Ci.mol⁻¹, thus the mCi to Ci range commonly used represents pmol to nmol quantities of fluoride.

**Radiochemical yield:** the amount of radiolabelled compounds divided by the initial amount of radioactive fluorine.

**Abbreviations**

<table><thead><tr><td>Abbreviation</td><td>Meaning</td></tr></thead><tbody><tr><td>ACN</td><td>Acetonitrile</td></tr><tr><td>AMBF<sub>3</sub></td><td>Alkylammoniomethyl-trifluoroborate</td></tr><tr><td>APM</td><td>N-(3-amino-propyl) methacrylamide hydrochloride</td></tr><tr><td>AT1</td><td>Angiotensin II receptor type 1</td></tr><tr><td>BFCA</td><td>Bifunctional chelating agent</td></tr><tr><td>BHT</td><td>Butylated hydroxytoluene</td></tr><tr><td>BSA</td><td>Bovine serum albumin</td></tr><tr><td>c.m.c</td><td>Critical micellar concentration</td></tr><tr><td>CaP</td><td>Calcium phosphonate</td></tr><tr><td>CDI</td><td>Carbonyldiimidazole</td></tr><tr><td>CLIO</td><td>Cross-Linked Iron Oxide</td></tr><tr><td>CT</td><td>X-ray computed tomography</td></tr><tr><td>CuAAC</td><td>Copper catalyzed alkyne-azide cycloaddtion</td></tr><tr><td>DBCO</td><td>Dibenzocyclooctyne</td></tr><tr><td>DCNP</td><td>Down-conversion nanoparticle</td></tr><tr><td>DLS</td><td>Dynamic light scattering</td></tr><tr><td>DMEM</td><td>Dulbecco's Modified Eagle Medium</td></tr><tr><td>DMSO</td><td>Dimethyl Sulfoxide</td></tr><tr><td>DOPC</td><td>1,2-dioleoyl-sn-glycero-3-phosphatidylcholine</td></tr><tr><td>DOPC</td><td>dioleoyl-sn-glycero-3-phosphatidylcholine</td></tr><tr><td>DOTPA</td><td>1,2-dioleoyl-3-trimethylammonium-propane</td></tr><tr><td>DPPC</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine</td></tr><tr><td>DPPE-DTPA</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N diethylenetriaminepentaacetic acid</td></tr><tr><td>DSPC</td><td>Distearoylphosphatidylcholine</td></tr><tr><td>DSPE</td><td>Distearoylphosphatidylethanolamine</td></tr><tr><td>EPR effect</td><td>Enhanced Permeability and Retention effect</td></tr><tr><td>FBBA</td><td>Fluorobenzyl-2-bromoacetamide</td></tr><tr><td>FBEM</td><td>4-fluorobenzamido-N-ethylmaleimide</td></tr><tr><td>FDG</td><td>Fluorodéoxyglucose</td></tr><tr><td>FDP</td><td>Fluorodipalmitin</td></tr><tr><td>FDPG</td><td>Fluoro-1,2-dipalmitoylglycerol</td></tr><tr><td>FEA</td><td>2-fluoroethyl azide</td></tr><tr><td>FMT</td><td>Fluorescence molecular tomography</td></tr><tr><td>FPyME</td><td>(2-fluoropyrid-3-yloxy)propylmaleimide</td></tr><tr><td>FSB</td><td>N-succinimidyl 4-fluorobenzoate</td></tr></tbody></table>
labelled through the grant EUR CARe N°ANR-18-EURE-0003 in the framework of the Programme des Investissements d'Avenir.

**Conflicts of Interest:** The authors declare no conflicts of interest.

**Nomenclature**

**Radiochemical Purity (RCP):** the proportion of the total radioactivity in the sample that is present as the desired radiolabelled species (definition from the International Atomic Energy Agency).

**Molar radioactivity:** the ratio of the amount of radiofluorinated, [¹⁸F]-containing, to the amount of fluorinated, ¹⁹F and ¹⁸F containing, compound.

**Specific radioactivity:** the activity per unit mass of a radionuclide is a physical property of the specified radionuclide and is given by the equation: $a[Bq/g] = \frac{N_A \ln 2}{M_{t1/2}}$, where N<sub>A</sub> is the Avogadro constant, M is the molar mass of the nuclide, and t<sub>1/2</sub> the half-life time of the radionuclide. For ¹⁸F, a = 1.71 × 10⁹ Ci.mol⁻¹, thus the mCi to Ci range commonly used represents pmol to nmol quantities of fluoride.

**Radiochemical yield:** the amount of radiolabelled compounds divided by the initial amount of radioactive fluorine.

**Abbreviations**

<table><thead><tr><td>Abbreviation</td><td>Meaning</td></tr></thead><tbody><tr><td>ACN</td><td>Acetonitrile</td></tr><tr><td>AMBF<sub>3</sub></td><td>Alkylammoniomethyl-trifluoroborate</td></tr><tr><td>APM</td><td>N-(3-amino-propyl) methacrylamide hydrochloride</td></tr><tr><td>AT1</td><td>Angiotensin II receptor type 1</td></tr><tr><td>BFCA</td><td>Bifunctional chelating agent</td></tr><tr><td>BHT</td><td>Butylated hydroxytoluene</td></tr><tr><td>BSA</td><td>Bovine serum albumin</td></tr><tr><td>c.m.c</td><td>Critical micellar concentration</td></tr><tr><td>CaP</td><td>Calcium phosphonate</td></tr><tr><td>CDI</td><td>Carbonyldiimidazole</td></tr><tr><td>CLIO</td><td>Cross-Linked Iron Oxide</td></tr><tr><td>CT</td><td>X-ray computed tomography</td></tr><tr><td>CuAAC</td><td>Copper catalyzed alkyne-azide cycloaddtion</td></tr><tr><td>DBCO</td><td>Dibenzocyclooctyne</td></tr><tr><td>DCNP</td><td>Down-conversion nanoparticle</td></tr><tr><td>DLS</td><td>Dynamic light scattering</td></tr><tr><td>DMEM</td><td>Dulbecco's Modified Eagle Medium</td></tr><tr><td>DMSO</td><td>Dimethyl Sulfoxide</td></tr><tr><td>DOPC</td><td>1,2-dioleoyl-sn-glycero-3-phosphatidylcholine</td></tr><tr><td>DOPC</td><td>dioleoyl-sn-glycero-3-phosphatidylcholine</td></tr><tr><td>DOTPA</td><td>1,2-dioleoyl-3-trimethylammonium-propane</td></tr><tr><td>DPPC</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine</td></tr><tr><td>DPPE-DTPA</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N diethylenetriaminepentaacetic acid</td></tr><tr><td>DSPC</td><td>Distearoylphosphatidylcholine</td></tr><tr><td>DSPE</td><td>Distearoylphosphatidylethanolamine</td></tr><tr><td>EPR effect</td><td>Enhanced Permeability and Retention effect</td></tr><tr><td>FBBA</td><td>Fluorobenzyl-2-bromoacetamide</td></tr><tr><td>FBEM</td><td>4-fluorobenzamido-N-ethylmaleimide</td></tr><tr><td>FDG</td><td>Fluorodéoxyglucose</td></tr><tr><td>FDP</td><td>Fluorodipalmitin</td></tr><tr><td>FDPG</td><td>Fluoro-1,2-dipalmitoylglycerol</td></tr><tr><td>FEA</td><td>2-fluoroethyl azide</td></tr><tr><td>FMT</td><td>Fluorescence molecular tomography</td></tr><tr><td>FPyME</td><td>(2-fluoropyrid-3-yloxy)propylmaleimide</td></tr><tr><td>FSB</td><td>N-succinimidyl 4-fluorobenzoate</td></tr></tbody></table>
<table>
  <tr>
    <td>FT-IR</td>
    <td>Fourier-transform infrared spectroscopy</td>
  </tr>
  <tr>
    <td>FTP</td>
    <td>Fluorothiophosphate</td>
  </tr>
  <tr>
    <td>GO</td>
    <td>Graphene oxide</td>
  </tr>
  <tr>
    <td>GRPR</td>
    <td>Gastrin-releasing peptide receptor</td>
  </tr>
  <tr>
    <td>GSH</td>
    <td>Glutathione</td>
  </tr>
  <tr>
    <td>HA</td>
    <td>Hydroxyapatite</td>
  </tr>
  <tr>
    <td>hbPG</td>
    <td>Hyperbranched polyglycerol</td>
  </tr>
  <tr>
    <td>HEPES</td>
    <td>N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid</td>
  </tr>
  <tr>
    <td>HFBII</td>
    <td>Class II hydrophobin</td>
  </tr>
  <tr>
    <td>HPLC</td>
    <td>High-performance liquid chromatography</td>
  </tr>
  <tr>
    <td>HPMA</td>
    <td>N-(2-hydroxypropyl)-methacrylamide</td>
  </tr>
  <tr>
    <td>HSA</td>
    <td>Human serum albumin</td>
  </tr>
  <tr>
    <td>i.v.</td>
    <td>Intravenous</td>
  </tr>
  <tr>
    <td>ICP-MS</td>
    <td>Inductively coupled plasma mass spectrometry</td>
  </tr>
  <tr>
    <td>ID</td>
    <td>Injected dose</td>
  </tr>
  <tr>
    <td>IONP</td>
    <td>Iron oxide nanoparticle</td>
  </tr>
  <tr>
    <td>K<sub>2.2.2</sub></td>
    <td>Kryptofix® 222</td>
  </tr>
  <tr>
    <td>LAH</td>
    <td>Lithium aluminium hydride</td>
  </tr>
  <tr>
    <td>LEH</td>
    <td>Liposome-encapsulating hemoglobin</td>
  </tr>
  <tr>
    <td>LMA</td>
    <td>Lauryl methacrylate</td>
  </tr>
  <tr>
    <td>mal-PEG-PE</td>
    <td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt</td>
  </tr>
  <tr>
    <td>MNP</td>
    <td>Magnetic nanoparticle</td>
  </tr>
  <tr>
    <td>MPC</td>
    <td>2-methacryloyloxyethyl phosphorylcholine</td>
  </tr>
  <tr>
    <td>MRI</td>
    <td>Magnetic resonance imaging</td>
  </tr>
  <tr>
    <td>MSC</td>
    <td>Mesenchymal stem cell</td>
  </tr>
  <tr>
    <td>MSNP</td>
    <td>Mesoporous silica nanoparticle</td>
  </tr>
  <tr>
    <td>MTDH</td>
    <td>Metadherin</td>
  </tr>
  <tr>
    <td>MWCO</td>
    <td>Molecular weight cut-off</td>
  </tr>
  <tr>
    <td>NFP</td>
    <td>4-nitrophenyl 2-[<sup>18</sup>F]fluoropropionate</td>
  </tr>
  <tr>
    <td>NFP</td>
    <td>Fluoropropionate p-nitrophenol</td>
  </tr>
  <tr>
    <td>nGO</td>
    <td>Nano-graphene oxide</td>
  </tr>
  <tr>
    <td>NIR</td>
    <td>Near-infrared</td>
  </tr>
  <tr>
    <td>NIRF</td>
    <td>Near-infrared fluorescence</td>
  </tr>
  <tr>
    <td>NMR</td>
    <td>Nuclear magnetic resonance</td>
  </tr>
  <tr>
    <td>NOTA</td>
    <td>2-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid</td>
  </tr>
  <tr>
    <td>NP</td>
    <td>Nanoparticle</td>
  </tr>
  <tr>
    <td>NPB4</td>
    <td>Fluorobenzylamide-poly(ethylene glycol)<sub>4</sub>-biotin</td>
  </tr>
  <tr>
    <td>PAMAM</td>
    <td>Poly(amido amine)</td>
  </tr>
  <tr>
    <td>PBS</td>
    <td>Phosphate-buffered saline</td>
  </tr>
  <tr>
    <td>PDI</td>
    <td>Polydispersity indexes</td>
  </tr>
  <tr>
    <td>PDT</td>
    <td>Photodynamic therapy</td>
  </tr>
  <tr>
    <td>PEG</td>
    <td>Polyethylene glycol</td>
  </tr>
  <tr>
    <td>PEG-DSPE</td>
    <td>N-[monomethoxy-polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine</td>
  </tr>
  <tr>
    <td>PET</td>
    <td>Positron emission tomography</td>
  </tr>
  <tr>
    <td>PFC</td>
    <td>Perfluorocabon</td>
  </tr>
  <tr>
    <td>PFCD</td>
    <td>Perfluorocarbon droplets</td>
  </tr>
  <tr>
    <td>PG</td>
    <td>Polyglycerol</td>
  </tr>
  <tr>
    <td>pHLIP</td>
    <td>pH (Low) Insertion Peptide</td>
  </tr>
  <tr>
    <td>pHPMA</td>
    <td>Poly-N-(2-hydroxypropyl) methacrylamide</td>
  </tr>
  <tr>
    <td>PLA</td>
    <td>Polylactic acid</td>
  </tr>
  <tr>
    <td>PLGA</td>
    <td>Poly(lactic-co-glycolic acid)</td>
  </tr>
  <tr>
    <td>PLL</td>
    <td>Polylysine</td>
  </tr>
  <tr>
    <td>pLMA</td>
    <td>Poly(lauryl methacrylate)</td>
  </tr>
  <tr>
    <td>POX</td>
    <td>Polyoxazolines</td>
  </tr>
  <tr>
    <td>PS</td>
    <td>Polysarcosine</td>
  </tr>
  <tr>
    <td>QD</td>
    <td>Quantum dot</td>
  </tr>
  <tr>
    <td>QMA</td>
    <td>Quaternary Methylamine resin</td>
  </tr>
  <tr>
    <td>RCP</td>
    <td>Radiochemical purity</td>
  </tr>
</table>
<table>
  <tr>
    <td>FT-IR</td>
    <td>Fourier-transform infrared spectroscopy</td>
  </tr>
  <tr>
    <td>FTP</td>
    <td>Fluorothiophosphate</td>
  </tr>
  <tr>
    <td>GO</td>
    <td>Graphene oxide</td>
  </tr>
  <tr>
    <td>GRPR</td>
    <td>Gastrin-releasing peptide receptor</td>
  </tr>
  <tr>
    <td>GSH</td>
    <td>Glutathione</td>
  </tr>
  <tr>
    <td>HA</td>
    <td>Hydroxyapatite</td>
  </tr>
  <tr>
    <td>hbPG</td>
    <td>Hyperbranched polyglycerol</td>
  </tr>
  <tr>
    <td>HEPES</td>
    <td>N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid</td>
  </tr>
  <tr>
    <td>HFBII</td>
    <td>Class II hydrophobin</td>
  </tr>
  <tr>
    <td>HPLC</td>
    <td>High-performance liquid chromatography</td>
  </tr>
  <tr>
    <td>HPMA</td>
    <td>N-(2-hydroxypropyl)-methacrylamide</td>
  </tr>
  <tr>
    <td>HSA</td>
    <td>Human serum albumin</td>
  </tr>
  <tr>
    <td>i.v.</td>
    <td>Intravenous</td>
  </tr>
  <tr>
    <td>ICP-MS</td>
    <td>Inductively coupled plasma mass spectrometry</td>
  </tr>
  <tr>
    <td>ID</td>
    <td>Injected dose</td>
  </tr>
  <tr>
    <td>IONP</td>
    <td>Iron oxide nanoparticle</td>
  </tr>
  <tr>
    <td>K<sub>2.2.2</sub></td>
    <td>Kryptofix® 222</td>
  </tr>
  <tr>
    <td>LAH</td>
    <td>Lithium aluminium hydride</td>
  </tr>
  <tr>
    <td>LEH</td>
    <td>Liposome-encapsulating hemoglobin</td>
  </tr>
  <tr>
    <td>LMA</td>
    <td>Lauryl methacrylate</td>
  </tr>
  <tr>
    <td>mal-PEG-PE</td>
    <td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt</td>
  </tr>
  <tr>
    <td>MNP</td>
    <td>Magnetic nanoparticle</td>
  </tr>
  <tr>
    <td>MPC</td>
    <td>2-methacryloyloxyethyl phosphorylcholine</td>
  </tr>
  <tr>
    <td>MRI</td>
    <td>Magnetic resonance imaging</td>
  </tr>
  <tr>
    <td>MSC</td>
    <td>Mesenchymal stem cell</td>
  </tr>
  <tr>
    <td>MSNP</td>
    <td>Mesoporous silica nanoparticle</td>
  </tr>
  <tr>
    <td>MTDH</td>
    <td>Metadherin</td>
  </tr>
  <tr>
    <td>MWCO</td>
    <td>Molecular weight cut-off</td>
  </tr>
  <tr>
    <td>NFP</td>
    <td>4-nitrophenyl 2-[<sup>18</sup>F]fluoropropionate</td>
  </tr>
  <tr>
    <td>NFP</td>
    <td>Fluoropropionate p-nitrophenol</td>
  </tr>
  <tr>
    <td>nGO</td>
    <td>Nano-graphene oxide</td>
  </tr>
  <tr>
    <td>NIR</td>
    <td>Near-infrared</td>
  </tr>
  <tr>
    <td>NIRF</td>
    <td>Near-infrared fluorescence</td>
  </tr>
  <tr>
    <td>NMR</td>
    <td>Nuclear magnetic resonance</td>
  </tr>
  <tr>
    <td>NOTA</td>
    <td>2-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid</td>
  </tr>
  <tr>
    <td>NP</td>
    <td>Nanoparticle</td>
  </tr>
  <tr>
    <td>NPB4</td>
    <td>Fluorobenzylamide-poly(ethylene glycol)<sub>4</sub>-biotin</td>
  </tr>
  <tr>
    <td>PAMAM</td>
    <td>Poly(amido amine)</td>
  </tr>
  <tr>
    <td>PBS</td>
    <td>Phosphate-buffered saline</td>
  </tr>
  <tr>
    <td>PDI</td>
    <td>Polydispersity indexes</td>
  </tr>
  <tr>
    <td>PDT</td>
    <td>Photodynamic therapy</td>
  </tr>
  <tr>
    <td>PEG</td>
    <td>Polyethylene glycol</td>
  </tr>
  <tr>
    <td>PEG-DSPE</td>
    <td>N-[monomethoxy-polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine</td>
  </tr>
  <tr>
    <td>PET</td>
    <td>Positron emission tomography</td>
  </tr>
  <tr>
    <td>PFC</td>
    <td>Perfluorocabon</td>
  </tr>
  <tr>
    <td>PFCD</td>
    <td>Perfluorocarbon droplets</td>
  </tr>
  <tr>
    <td>PG</td>
    <td>Polyglycerol</td>
  </tr>
  <tr>
    <td>pHLIP</td>
    <td>pH (Low) Insertion Peptide</td>
  </tr>
  <tr>
    <td>pHPMA</td>
    <td>Poly-N-(2-hydroxypropyl) methacrylamide</td>
  </tr>
  <tr>
    <td>PLA</td>
    <td>Polylactic acid</td>
  </tr>
  <tr>
    <td>PLGA</td>
    <td>Poly(lactic-co-glycolic acid)</td>
  </tr>
  <tr>
    <td>PLL</td>
    <td>Polylysine</td>
  </tr>
  <tr>
    <td>pLMA</td>
    <td>Poly(lauryl methacrylate)</td>
  </tr>
  <tr>
    <td>POX</td>
    <td>Polyoxazolines</td>
  </tr>
  <tr>
    <td>PS</td>
    <td>Polysarcosine</td>
  </tr>
  <tr>
    <td>QD</td>
    <td>Quantum dot</td>
  </tr>
  <tr>
    <td>QMA</td>
    <td>Quaternary Methylamine resin</td>
  </tr>
  <tr>
    <td>RCP</td>
    <td>Radiochemical purity</td>
  </tr>
</table>
<table><tr><td>RCY</td><td>Radiochemical yield</td></tr><tr><td>RE</td><td>Rare earth nanoparticle</td></tr><tr><td>ROMP</td><td>Ring-opening metathesis polymerization</td></tr><tr><td>ROP</td><td>Ring-opening polymerization</td></tr><tr><td>rTLC</td><td>Radio thin-layer chromatography</td></tr><tr><td>SEC</td><td>Size-exclusion chromatography</td></tr><tr><td>SFB</td><td>N-succinimidyl 4-[<sup>18</sup>F]fluorobenzoate</td></tr><tr><td>SiFA</td><td>Silicon-fluoride-acceptor</td></tr><tr><td>SiRNA</td><td>Small interfering RNA</td></tr><tr><td>SLNC</td><td>Solid lipid nanocomposite</td></tr><tr><td>SN</td><td>Sphingomyelin nanometric emulsions</td></tr><tr><td>S<sub>N</sub>2</td><td>Nucleophilic substitution second order</td></tr><tr><td>S<sub>N</sub>Ar</td><td>Nucleophilic aromatic substitution</td></tr><tr><td>SPAAC</td><td>Strain-promoted alkyne-azide cycloaddition</td></tr><tr><td>SPANC</td><td>Strain promoted alkyne-nitrone cycloaddition</td></tr><tr><td>SPECT</td><td>Single-photon emission computed tomography</td></tr><tr><td>SUV</td><td>Standardized uptake value</td></tr><tr><td>TATE</td><td>Octreotate</td></tr><tr><td>TCO</td><td>Trans-cyclooctene</td></tr><tr><td>TCPSi</td><td>Thermally carbonized porous silicon</td></tr><tr><td>TEG</td><td>Triethyleneglycol</td></tr><tr><td>TEM</td><td>Transmission electron microscopy</td></tr><tr><td>TFA</td><td>Trifluoroacetic acid</td></tr><tr><td>TGA</td><td>Thermogravimetric analysis</td></tr><tr><td>THCPSi</td><td>Thermally hydrocarbonized porous silicon</td></tr><tr><td>TLC</td><td>Thin-layer chromatography</td></tr><tr><td>TOPSi</td><td>Thermally oxidized porous silicon</td></tr><tr><td>UC</td><td>Up-conversion</td></tr><tr><td>UCNP</td><td>Up-conversion nanoparticle</td></tr><tr><td>USPIO</td><td>Ultrasmall superparamagnetic iron oxide</td></tr><tr><td>UV-Vis</td><td>Ultraviolet-visible spectroscopy</td></tr><tr><td>XPS</td><td>X-ray photoelectron spectroscopy</td></tr><tr><td>XRD</td><td>X-ray diffraction</td></tr></table>

References

1. Sharifi, M.; Cho, W.C.; Ansariesfahani, A.; Tarharoudi, R.; Malekisarvar, H.; Sari, S.; Bloukh, S.H.; Edis, Z.; Amin, M.; Gleghorn, J.P.; et al. An Updated Review on EPR-Based Solid Tumor Targeting Nanocarriers for Cancer Treatment. *Cancers* **2022**, *14*, 2868. [CrossRef]

2. Wu, J. The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application. *J. Pers. Med.* **2021**, *11*, 771. [CrossRef]

3. Baker, M. The Whole Picture. *Nature* **2010**, *463*, 977–979. [CrossRef]

4. Brandt, M.; Cardinale, J.; Aulsebrook, M.L.; Gasser, G.; Mindt, T.L. An Overview of PET Radiochemistry, Part 2: Radiometals. *J. Nucl. Med.* **2018**, *59*, 1500–1506. [CrossRef] [PubMed]

5. Pichler, V.; Berroterán-Infante, N.; Philippe, C.; Vraka, C.; Klebermass, E.-M.; Balber, T.; Pfaff, S.; Nics, L.; Mitterhauser, M.; Wadsak, W. An Overview of PET Radiochemistry, Part 1: The Covalent Labels <sup>18</sup>F, <sup>11</sup>C, and <sup>13</sup>N. *J. Nucl. Med.* **2018**, *59*, 1350–1354. [CrossRef] [PubMed]

6. Zhang, C.; Yan, K.; Fu, C.; Peng, H.; Hawker, C.J.; Whittaker, A.K. Biological Utility of Fluorinated Compounds: From Materials Design to Molecular Imaging, Therapeutics and Environmental Remediation. *Chem. Rev.* **2022**, *122*, 167–208. [CrossRef]

7. Liao, A.-H.; Wu, S.-Y.; Wang, H.-E.; Weng, C.-H.; Wu, M.-F.; Li, P.-C. Evaluation of <sup>18</sup>F-Labeled Targeted Perfluorocarbon-Filled Albumin Microbubbles as a Probe for microUS and microPET in Tumor-Bearing Mice. *Ultrasonics* **2013**, *53*, 320–327. [CrossRef] [PubMed]

8. Yang, Y.; Liu, Y.; Jiang, Y. Recent Advances in Perfluorocarbon-Based Delivery Systems for Cancer Theranostics. *Mol. Pharm.* **2023**, *20*, 3254–3277. [CrossRef]

9. Wu, L.; Liu, F.; Liu, S.; Xu, X.; Liu, Z.; Sun, X. Perfluorocarbons-Based <sup>19</sup>F Magnetic Resonance Imaging in Biomedicine. *Int. J. Nanomed.* **2020**, *15*, 7377–7395. [CrossRef]

10. Amir, N.; Green, D.; Kent, J.; Xiang, Y.; Gorelikov, I.; Seo, M.; Blacker, M.; Janzen, N.; Czorny, S.; Valliant, J.F.; et al. <sup>18</sup>F-Labeled Perfluorocarbon Droplets for Positron Emission Tomography Imaging. *Nucl. Med. Biol.* **2017**, *54*, 27–33. [CrossRef]
<table><tr><td>RCY</td><td>Radiochemical yield</td></tr><tr><td>RE</td><td>Rare earth nanoparticle</td></tr><tr><td>ROMP</td><td>Ring-opening metathesis polymerization</td></tr><tr><td>ROP</td><td>Ring-opening polymerization</td></tr><tr><td>rTLC</td><td>Radio thin-layer chromatography</td></tr><tr><td>SEC</td><td>Size-exclusion chromatography</td></tr><tr><td>SFB</td><td>N-succinimidyl 4-[<sup>18</sup>F]fluorobenzoate</td></tr><tr><td>SiFA</td><td>Silicon-fluoride-acceptor</td></tr><tr><td>SiRNA</td><td>Small interfering RNA</td></tr><tr><td>SLNC</td><td>Solid lipid nanocomposite</td></tr><tr><td>SN</td><td>Sphingomyelin nanometric emulsions</td></tr><tr><td>S<sub>N</sub>2</td><td>Nucleophilic substitution second order</td></tr><tr><td>S<sub>N</sub>Ar</td><td>Nucleophilic aromatic substitution</td></tr><tr><td>SPAAC</td><td>Strain-promoted alkyne-azide cycloaddition</td></tr><tr><td>SPANC</td><td>Strain promoted alkyne-nitrone cycloaddition</td></tr><tr><td>SPECT</td><td>Single-photon emission computed tomography</td></tr><tr><td>SUV</td><td>Standardized uptake value</td></tr><tr><td>TATE</td><td>Octreotate</td></tr><tr><td>TCO</td><td>Trans-cyclooctene</td></tr><tr><td>TCPSi</td><td>Thermally carbonized porous silicon</td></tr><tr><td>TEG</td><td>Triethyleneglycol</td></tr><tr><td>TEM</td><td>Transmission electron microscopy</td></tr><tr><td>TFA</td><td>Trifluoroacetic acid</td></tr><tr><td>TGA</td><td>Thermogravimetric analysis</td></tr><tr><td>THCPSi</td><td>Thermally hydrocarbonized porous silicon</td></tr><tr><td>TLC</td><td>Thin-layer chromatography</td></tr><tr><td>TOPSi</td><td>Thermally oxidized porous silicon</td></tr><tr><td>UC</td><td>Up-conversion</td></tr><tr><td>UCNP</td><td>Up-conversion nanoparticle</td></tr><tr><td>USPIO</td><td>Ultrasmall superparamagnetic iron oxide</td></tr><tr><td>UV-Vis</td><td>Ultraviolet-visible spectroscopy</td></tr><tr><td>XPS</td><td>X-ray photoelectron spectroscopy</td></tr><tr><td>XRD</td><td>X-ray diffraction</td></tr></table>

References

1. Sharifi, M.; Cho, W.C.; Ansariesfahani, A.; Tarharoudi, R.; Malekisarvar, H.; Sari, S.; Bloukh, S.H.; Edis, Z.; Amin, M.; Gleghorn, J.P.; et al. An Updated Review on EPR-Based Solid Tumor Targeting Nanocarriers for Cancer Treatment. *Cancers* **2022**, *14*, 2868. [CrossRef]

2. Wu, J. The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application. *J. Pers. Med.* **2021**, *11*, 771. [CrossRef]

3. Baker, M. The Whole Picture. *Nature* **2010**, *463*, 977–979. [CrossRef]

4. Brandt, M.; Cardinale, J.; Aulsebrook, M.L.; Gasser, G.; Mindt, T.L. An Overview of PET Radiochemistry, Part 2: Radiometals. *J. Nucl. Med.* **2018**, *59*, 1500–1506. [CrossRef] [PubMed]

5. Pichler, V.; Berroterán-Infante, N.; Philippe, C.; Vraka, C.; Klebermass, E.-M.; Balber, T.; Pfaff, S.; Nics, L.; Mitterhauser, M.; Wadsak, W. An Overview of PET Radiochemistry, Part 1: The Covalent Labels <sup>18</sup>F, <sup>11</sup>C, and <sup>13</sup>N. *J. Nucl. Med.* **2018**, *59*, 1350–1354. [CrossRef] [PubMed]

6. Zhang, C.; Yan, K.; Fu, C.; Peng, H.; Hawker, C.J.; Whittaker, A.K. Biological Utility of Fluorinated Compounds: From Materials Design to Molecular Imaging, Therapeutics and Environmental Remediation. *Chem. Rev.* **2022**, *122*, 167–208. [CrossRef]

7. Liao, A.-H.; Wu, S.-Y.; Wang, H.-E.; Weng, C.-H.; Wu, M.-F.; Li, P.-C. Evaluation of <sup>18</sup>F-Labeled Targeted Perfluorocarbon-Filled Albumin Microbubbles as a Probe for microUS and microPET in Tumor-Bearing Mice. *Ultrasonics* **2013**, *53*, 320–327. [CrossRef] [PubMed]

8. Yang, Y.; Liu, Y.; Jiang, Y. Recent Advances in Perfluorocarbon-Based Delivery Systems for Cancer Theranostics. *Mol. Pharm.* **2023**, *20*, 3254–3277. [CrossRef]

9. Wu, L.; Liu, F.; Liu, S.; Xu, X.; Liu, Z.; Sun, X. Perfluorocarbons-Based <sup>19</sup>F Magnetic Resonance Imaging in Biomedicine. *Int. J. Nanomed.* **2020**, *15*, 7377–7395. [CrossRef]

10. Amir, N.; Green, D.; Kent, J.; Xiang, Y.; Gorelikov, I.; Seo, M.; Blacker, M.; Janzen, N.; Czorny, S.; Valliant, J.F.; et al. <sup>18</sup>F-Labeled Perfluorocarbon Droplets for Positron Emission Tomography Imaging. *Nucl. Med. Biol.* **2017**, *54*, 27–33. [CrossRef]