Methods for Radiolabeling Nanoparticles (Part 3): Therapeutic Use

Following previously published systematic reviews on the diagnostic use of nanoparticles (NPs), in this manuscript, we report published methods for radiolabeling nanoparticles with therapeutic alpha-emitting, beta-emitting, or Auger’s electron-emitting isotopes. After analyzing 234 papers, we found that different methods were used with the same isotope and the same type of nanoparticle. The most common type of nanoparticles used are the PLGA and PAMAM nanoparticles, and the most commonly used therapeutic isotope is 177Lu. Regarding labeling methods, the direct encapsulation of the isotope resulted in the most reliable and reproducible technique. Radiolabeled nanoparticles show promising results in metastatic breast and lung cancer, although this field of research needs more clinical studies, mainly on the comparison of nanoparticles with chemotherapy.


Introduction
In the last ten years, the field of nanomedicine and associated research has enormously expanded, without a significant reduction due to the COVID pandemic in the previous three years.
This exponential growth in publications is particularly evident if we use "nanoparticles" as a search input, rising from 17,046 in 2012 to 34,132 in 2022 (Figure 1).

Radiolabeling of NPs with Auger's Electron-Emitting Isotopes
Auger's electrons are emitted from radioactive nuclei due to the Auger effect.The Auger effect is the emission of an Auger's electron from an atom that has gained energy by filling an external electronic shell, which fills a vacancy in an external electronic orbital that can occur during radioactive decay.A few of these publications discuss the use of "radiolabeled nanoparticles", which include "radiolabeled nanoparticles for diagnostic SPECT use", "radiolabeled nanoparticles for diagnostic PET use", and "radiolabeled nanoparticles for therapy" (Figure 1).
In general, the methods for NP measurement in biological samples depend on the chemical composition and structure of NPs, and there is no universal technique that is suitable for all NPs.Among the techniques established for NP measurement and imaging, isotope tracing, particularly radioactive tracing, is one of the most powerful for assigning a source and monitoring its distribution from the nano to the global scale.Furthermore, radiolabeled NPs have received significant attention in nuclear medicine, molecular imaging, drug delivery, and radiation therapy [1,2].
We previously published two systematic reviews on "methods for radiolabeling nanoparticles for diagnostic SPECT use" [3] and "methods for radiolabeling nanoparticles for diagnostic PET use" [4].This paper addresses a systematic review of the published research on "methods for radiolabeling nanoparticles for therapy", including an Auger's electron-emitting, Alpha-emitting, and Beta-emitting isotopes.

Radiolabeling of NPs with Auger's Electron-Emitting Isotopes
Auger's electrons are emitted from radioactive nuclei due to the Auger effect.The Auger effect is the emission of an Auger's electron from an atom that has gained energy by filling an external electronic shell, which fills a vacancy in an external electronic orbital that can occur during radioactive decay.
Auger's electrons have a high level of linear energy transfer (LET) (1-26 keV mm −1 ) and a very small particle range (<0.5 mm).These properties enable the radioisotope to be transported intracellularly to the nucleus, maximizing cytotoxic activity, which results in DNA double-strand breaks [5].
Several isotopes can emit Auger's electrons, such as 64 Cu, 161 Tb, 195m Pt, 111 In, 67 Ga, 99m Tc, 125 I, 124 I, and 123 I.However, most of these isotopes are used for diagnostic purposes and have already been described in the Part 1 and Part 2 reviews [3,4].
Here, we describe the labeling methods using 64 Cu, one of the most commonly used radioisotopes for therapeutic purposes, as summarized in Table 1.Tested on keratinforming tumor cell line HeLa [15] n.d.a.= no data available; NPs = nanoparticles.

Direct Radiolabeling
The 64 Cu has been used to directly and indirectly label different NPs.Copper-sulfide nanoparticles (Cu 2 S NPs) are among the NPs that can be labeled by the direct method and show therapeutic effects.The synthesis of these NPs begins with the introduction of Na 2 S into an aqueous solution of CuCl 2 together with an aliquot of 64 CuCl 2 , in the presence of PEG-SH.The rapidity of this method, which takes only 20 min, is important for minimizing radioactive decay.The radiolabeling efficiency and stability were analyzed using instant thin-layer chromatography (iTLC), and the results showed that more than 99% of the radioactivity was associated with the radiolabeled NPs at the end of synthesis [6].
Gailkwad et al. proposed another strategy for direct radiolabeling with NPs.Chitosan (CHS) was first radiolabeled and then converted to NPs through ionotropic gelation.The authors tried this method with several therapeutic radioisotopes, such as 64 Cu, 90 Y, 166 Ho, and 177 Lu.The radiolabeling process started with the mixing of the CHS solution with a water-soluble chitosan (COS) solution, and, after adjusting the pH to 5 using NaOH, the solution was stirred for 30 min at room temperature.Subsequently, the relevant radiometal solution was added with TPP and stirred for 10 min, producing radioactive-CH NPs.The chitosan NPs were cross-linked with a glutaraldehyde solution while stirring for at least 30 min.The radiochemical purity was determined using iTLC and was greater than 98% for all the samples.This novel technique takes advantage of chitosan's unique physical and chemical properties to provide an easy, rapid, and highly selective radiolabeling strategy [7].
Instead of chitosan, other organic materials can also be used for the synthesis of radiolabeled nanoparticles; Zhou et al. fabricated radiolabeled melanin NPs (MNPs) with a chelator-free approach, mixed them with 64 CuCl 2 in the presence of a sodium acetate (NaOAc) buffer, and left the mixture to incubate for 1 h at 40 • C.An Al aliquot of trifluoroacetic acid (TFA) was used to stop the reaction.
The radiochemical purity (RCP) was evaluated by ITLC, and it was found to be greater than 95%.The therapeutic effect was evaluated on human-epidermoid-carcinoma-tumorbearing nude mice, and the findings showed a positive result, in that the tumor growth was significantly inhibited after treatment with a single dose (55.5 MBq).The comprehensive study concluded that 64 Cu-MNPs is a promising candidate for cancer therapy [8].
Gholami et al. developed radiolabeled NPs with superparamagnetic iron oxide (SPIO) cores, commonly known as Feraheme ® (FH) NPs, in which radioisotopes bind directly to the surface of the SPIO core.The therapeutic radioisotopes used were 223 Ra, 177 Lu, 90 Y, and 64 Cu.The study showed that the FH NPs could be radiolabeled with high RCY and RCP using both diagnostic radioisotopes with long-range beta emitters and therapeutic radioisotopes with short-range alpha emitters [9].
Boron nitride nanotubes (BNNTs) are cylindrical structures formed by atoms of boron (B) and nitrogen (N).The empty internal spaces allow BNNTs to occupy various chemical species, including radioisotopes, in their vacant sites.The solvothermal process is mainly used to radiolabel BNNTs with 64 Cu.In this procedure, the BNNTs were dispersed in anhydrous ethanol, and the 64 Cu was encapsulated using an autoclave with a polytetrafluoroethylene (PTFE) vessel for 2 h at 180 • C.This method resulted in a stable and contaminant-free radioisotope, and the TEM images showed that the BNNTs were structurally well organized, presenting 64 Cu nanoparticles not only in their internal channels but also on their surfaces [10].
All of these studies convey the fact that radiolabeling occurs during nanoparticle synthesis.However, Xie et al. studied a post-synthesis approach that allows the encapsulation of 64 Cu into NPs, particularly covellite nanocrystals (CuS NCs).The researchers demonstrated that the covellite NCs dispersed with toluene incorporated Cu(I) ions [11].Consequently, the in situ reduction of 64 Cu (II) to 64 Cu(I) was required.For this purpose, ascorbic acid, a nontoxic and mild reducing agent, was added to the solution.The reactions on the NCs were tested in an aqueous environment, and most of the incorporation process was complete within a few minutes at room temperature [12].
A new carrier system based on a simple single-step precipitation technique was designed to synthesize radioactive barium sulfate nanoparticles (BaSO 4 NPs) with excellent stability in an aqueous environment (<5% activity release).In this work, the incorporation of several radionuclides, such as 131 Ba, 133 Ba, 224 Ra, 18 F, 64 Cu, 89 Zr, 111 In, and 177 Lu, was investigated.The synthesis and radiolabeling process forecasts barium-alendronate precipitation by adding ethanol and the respective radionuclide, which provide nanotemplates for the subsequent growth of the alendronate-containing BaSO 4 NPs.The radio-chemical yield (RCY) was expressed as the ratio of the amount of activity in the NP pellets to the starting activity, and while the 177 Lu-BaSO 4 NPs had an acceptable RCY, of 69%, the 64 Cu-BaSO 4 NPs did not achieve sufficient labeling due to the affinity of the alendronate as a complexing agent for copper ions [13].
The neutron irradiation of the non-radioactive isotopes that comprise NPs is another method for obtaining radioactive NPs.The hydrothermal coprecipitation method was used to synthesize hydroxyapatite (HA) NPs with copper.The synthesis started with the introduction of two precursor solutions: the first was prepared by mixing in water cetyltrimethyl-ammonium-bromide (CTAB), calcium nitrate tetrahydrate (Ca(NO 3 ) 2 •4H 2 O,) and copper(II) nitrate hydrate (Cu(NO 3 ) 2 •3H 2 O).In contrast, the second solution was obtained by mixing potassium phosphate dibasic trihydrate (K 2 HPO 4 •3H 2 O) with water.After adjusting the pH of both solutions to 12, the precursor solution was left for 6 h, and then the second solution was added dropwise to the first solution.The final product was stirred continuously throughout the night before being transferred to an autoclave to continue the hydrothermal treatment at 100 • C for 10 h.The materials were calcined at 600 • C under air pressure for 6 h after centrifugation and vacuum filtering to remove the organic template and grow the tenorite phase.The NPs were irradiated for 2 h with a thermal neutron flow to activate the 64 Cu [14].

Indirect Radiolabeling
Most of the previous studies involved the use of 64 Cu for a diagnostic purpose, which we discussed in our previous review [3].However, there are few works in the literature on the indirect labeling of nanoparticles with 64 Cu for therapeutic purposes.
Rossin et al. studied folate-conjugated shell-cross-linked nanoparticles (SCKs) functionalized with 1,4,8,11-tetraazacyclotetradecane-N, N , N , and N -tetraacetic acid (TETA).This bifunctional chelator (BFC) is a macrocyclic ligand that provides thermodynamic sta-bility and copper-complex kinetics in vivo.To obtain these NPs, a multi-step process was required.The authors proceeded to synthesize the BFC after conjugating the SCK with folate, allowing them to react with a mono tert-Boc-protected diamine (N-butoxycarbonyl-[2,2 -(ethylenedioxy) bis(ethylamine)]) with TETA in the presence of EDC for 16 h.The tert-Boc-protecting group was cleaved using trifluoroacetic acid, and the TETA-NH 2 product was purified via methanol and acetone precipitation.The carboxylic groups on the SCK-folate shells were activated by the EDC-NHs system in a PBS solution for 2 h at 4 • C while stirring.The TETA-NH2 was added after the excess EDC-NHS was separated, and the reaction mixtures were gently stirred overnight at 4 • C. The 64 Cu chloride was converted to 64 Cu acetate for radiolabeling by adding an ammonium acetate buffer (pH 6.5) and added to the TETA-SCK solution.The solution was incubated in a thermomixer (1000 rpm) under mild reaction conditions (43 • C for 2.5 h), yielding radiolabeled NPs with a high RCP (>95%) [15].

Discussion
To summarize, many strategies can be used to achieve 64 Cu-labeled NPs by direct radiolabeling.The possibility of radiolabeling NPs without the use of BFCs is due to their chemical characteristics and, furthermore, this possibility does not apply to all types of NP.For indirect radiolabeling, only TETA was studied for this radioisotope.Since this is a multi-step process, the chemical characteristics of the NPs must not be modified, in order to limit the influence on their targeting specificity as far as possible.

Radiolabeling of NPs with Alpha-Emitting Isotopes
Alpha particles (helium nuclei) have a high LET, of approximately 80-100 keV/µm, and a penetration range that is typically <100 µm (equivalent to a few cells layer).Due to their high LET, alpha particles mostly interact with DNA, causing irreversible double-strand breaks, and other effects such as G2-phase delay, the generation of reactive oxygen species (ROS), and chromosomal rearrangements [17].Additionally, β-radiation, for example, can induce damage in neighboring cells [18].
The methods for radiolabeling NPs with α-emitting isotopes are summarized in Table 2.

Radiolabeling with Astatine-211
One of the most promising candidates for targeted α-radionuclide therapy (TAT) is 211 At.Its long half-life (7.2 h) allows distribution from the production site, labeling procedures, quality control (QC), and clinical application without producing long-lived daughters emitting other α or β particles.Furthermore, this radionuclide is easily produced by cyclotrons with the nuclear reaction of 209 Bi (α, 2n) 211 At, followed by dry distillation and isolation from irradiated bismuth [18].These characteristics make this radionuclide suitable for radiolabeling several types of NP.

Direct Radiolabeling
Astatine is commonly considered a halogen, but the aryl-carbon-halogen bond is very weak, and radiolabeling methods used for other halogens (i.e., iodine) cannot be used without rapid loss, in vivo, of 211 At from NPs.
To overcome this problem, several authors proposed the use of gold nanoparticles (AuNPs) as carriers for 211 At.The use of gold is based on the fact that iodine is absorbed by the surfaces of noble metals, establishing strong covalent bonds.Due to the similarities between iodine and astatine, it was expected that 211 At would form a covalent bond on the surfaces of the AuNPs.For the radiolabeling process, the 211 At was diluted in Na 2 SO 3 /methanol, the solution was evaporated, and the radionuclide was dissolved in water.For the labeling, 20 mL of 211 At were added to 200 mL of AuNPs, the pH was maintained at 6, and the final solution was incubated at room temperature under stirring.The radiolabeling yield was >99%, as evaluated by iTLC, with MeOH [19][20][21].The same method was used for the radiolabeling of gold nanostars (GNSs).In this case, centrifugation was used to separate the free 211 At from the 211 At-GNS at the end of the labeling procedure [22].
Kucka et al. investigated the feasibility of radiolabeling silver nanoparticles (AgNPs) using the affinity of astatine for metallic silver.In citrate-phosphate buffers, commercially available AgNPs were labeled at pH 5 and pH 8. Chloramine-T was used to oxidize the 211 At, followed by reduction with ascorbic acid.The results showed that the pH did not affect the labeling yield, which was greater than 95% in both cases.The same method was then applied to experimentally synthesized AgNPs using KMnO 4 as an oxidant and NaBH 4 or Na 2 SO 3 as a reducing agent.The radiolabeling yield was 77.1% without a reducing/oxidant agent, while rates of 53.7%, 99.7%, and 94.2% were achieved in the presence of KMnO 4 , NaBH 4 , and Na 2 SO 3 , respectively.The use of oxidating or reducing agents can overcome limitations such as the low reproducibility of the radiolabeling yields.The researchers found that astatine can oxidize due to water radiolysis and does not allow it to react on the surface of silver in an oxidation state.The radiolabeling yield and reproducibility can be increased by protecting astatine from oxidation.It was also shown that low water contents in methanol can quench radiolysis and cause it to behave like an oxygen-radical scavenger [23].

Discussion
So far, 211 At has been radiolabeled with NPs by the direct method only.It is yet to be explored with indirect and encapsulation methods.
The direct radiolabeling of NPs with 211 At is based on the affinity between the radionuclide and the NPs.The 211 At showed a high affinity for gold surfaces, while with silver surfaces, it is essential to prevent astatine oxidation by using a reducing agent in the radiolabeling process.

Radiolabeling with Actinium-225
Actinium-225 ( 225 Ac) acts as an α-particle generator because of its half-life of 10 days and multiple decaying properties ( 221 Fr, 217 At, 213 Bi) [41].The decaying of a radionuclide can occur via a cascade of six radionuclide daughters with short half-lives.This predominantly decaying path generates four α-particles with high energy, while the cascade releases two β disintegrations, such as 221 Fr and 213 Bi.The 213 Bi is an α and β emitter with a half-life of 46 min.It decays to 213 Po by emitting α particles with half-lives of 4.2 µs, breaking the DNA double strand and causing cytotoxic effects [42].

Direct Radiolabeling
Iron-oxide-based nanoparticles (SPIONs) have attracted broad interest for their intrinsic magnetic properties, like their high surface-area-to-volume ratio and their ability to change their core composition and surface chemically.This chemical modification can be performed through the direct labeling of radioisotopes.Cędrowska et al. synthetized NPs by co-precipitating FeCl 3 and 225 Ac, followed by the addition of ammonia to obtain a solution with pH 10.The formed solution was stirred for 15 min, reaching a labeling efficiency of >98% [24].
Ruggiero et al. conjugated DOTA with the primary amines of the SWNT sidewalls.After incubation, 225 Ac was added to the solution, producing radiolabeled NPs with a 96% RCP [29].
A DOTA-derivative chelator, TADOTAGA, was studied as a BFC for the radiolabeling of AuNPs.The labeling process was similar to that for DOTA.First, TADOTAGA was added to the gold salt solution while stirring, and then a NaBH 4 aqueous solution was added after a few minutes.After stirring for 1 h, followed by dialysis, the 225 AcCl 3 was added and kept in the incubator for 30 min at 70 • C. The sample purification was performed through centrifugation for 15 min at 8110 rpm.The percentage of 225 Ac was incorporated, and the RCP was determined by iTLC.The results showed RCY values of 86.0 ± 1.8% before centrifugation and 68.5 ± 2.3% after centrifugation, with the RCP greater than 93% [28].

Radiolabeling by Encapsulation
Sofou et al. used liposomes as therapeutic agents for micro metastases and selected the method of encapsulating the radionuclide within the NP.For the synthesis of radioactive liposomes, 225 Ac was mixed with sucrose-loaded liposomes in PBS and incubated at room temperature for 1h.The binding of the radionuclide to the liposomes was measured using an ultracentrifugation assay, 142,000× g for 2 h at 25 • C, allowing the separation of the labeled liposomes from the free 225 Ac [25].
Inorganic nanoparticles were used as vehicles for targeted radiotherapy.McLaughlin et al. performed a study in which they radiolabeled gold-coated lanthanide phosphate NPs ({La0.5 Gd0.5}( 225 Ac)PO 4 NPs) and LaPO 4 (monazite) NPs without BFCs.Firstly, 225 AcCl 3 was added to the lanthanide mixture, followed by four additional shells of pure GdPO 4 .The resulting mixture was sealed and heated for 3 h at 90 • C and, finally, purified overnight via dialysis.To test the retention of 225 Ac decay in vitro, an aliquot was taken from the dialysis tube at different time points and analyzed.The results showed that these NPs were very stable over three weeks, and the NPs retained more than 99.9% of the 225 Ac [26].
Woodward et al. synthesized LaPO 4 NPs.In this process, LaCl 3 in H 2 O was diluted with HCl containing 225 AcCl 3 .The solution was then evaporated, resuspended in tri-nbutyl phosphate and phenyl-ether, and heated to 100 • C under vacuum with stirring.Next, an aliquot of H 3 PO 4 solution was added, and the total mixture was heated to 200 • C in an argon atmosphere while stirring for 2 h.Once the solution reached room temperature, the product was separated from the colloids by adding a combination of hexane and hexadecane and centrifuging for 20 min at 14.000× g.A sample of NPs was dialyzed for one month to test the in vitro release of the radionuclides.The presence of free 221 Fr and 213 Bi was measured in the sample for 2 min at 1-min intervals, while the presence of 225 Ac was determined by recounting the same sample after 1 h.The results showed the partial sequestration of the 221 Fr and 213 Bi daughters, while the 225 Ac was completely retained in the NPs [27].
Mulvey et al. investigated the radiolabeling of SWCNT with 225 Ac by three techniques.In the first, 225 Ac 3+ ions were added to the NPs; in the second, a mixture of Gd 3+ ions and 225 Ac 3+ ions were added together; and in the third, Gd 3+ ions, followed by 225 Ac 3+ ions, were added sequentially.The samples were sonicated for 2 h to ensure the encapsulation of the 225 Ac, and then allowed to equilibrate overnight.All the samples were washed and filtered the next day with a paper membrane.The extent of the 225 Ac 3+ labeling was determined by serial washing via centrifuge filtration.For the samples with only 225 Ac 3+ , over 95% of the activity remained with the NPs, while for the other two techniques, only 50% of the activity remained associated, probably due to the excess of Gd 3+ ions saturating all the binding sites for the 225 Ac.Challenge experiments in human serum were conducted to simulate in vivo conditions, and the results showed that only 40% of the 225 Ac 3+ remained for the US tubes labeled with 225 Ac 3+ only [30].
Hernández-Jiménez et al. encapsulated 225 Ac-radionuclides into reconstituted highdensity lipoprotein (rHDL) nanoparticles.The findings demonstrated that the fabricated 13-nanometer nanosystem had more a rate of radiochemical purity of more than 99% and was stable in human serum [31].

Discussion
According to published methodologies, the most significant aspect of radiolabeled 225 Ac-NPs is the stability of the labeled compound over time.The stability of labeling is related not only to 225 Ac, but also to its decay products [43].For this reason, materials with high density are required to ensure the sufficient retention of all the decay products within the NPs to limit the cytotoxicity to healthy tissues.

Radiolabeling with Radium-223
As a radium dichloride ([ 223 Ra]RaCl 2 ), 223 Ra was the first α-emitting isotope with FDA marketing authorization for the treatment of bone metastases, and it was examined as a potential for targeted radionuclide therapy employing NPs as carriers.This isotope does not have the translocation problem, which is an advantage; while 225 Ac has a decay chain leading to the generation of daughter nuclides, 75% of the total alphas in the 223 Ra are delivered within a few seconds (T 1/2 = 4 s) [44][45][46].

Direct Radiolabeling
The fundamental restriction of 223 Ra in clinical applications is the difficulty in binding the radioisotope to biomolecules in a stable way.This can be overcome by direct radiolabeling or by encapsulating 223 Ra into NPs.
The encapsulation of this radioisotope in GdVO 4 NPs was performed by Toro-Gonzalez et al., with two different methods.The first consisted in mixing a solution containing 223 Ra evaporated to dryness using an infrared heatlamp and a hot plate with a GdCl 3 solution and stirred for 10 min, followed by the dropwise addition of a Na 3 VO 4 solution.In the second method, Na 3 VO 4 was mixed with the radionuclides, followed by the addition of the GdCl 3 solution dropwise.
Radiolabeled GdVO 4 NPs were dialyzed against water to assess the encapsulation and to evaluate the radiochemical yield of the radioisotope and the leakage of the decay daughters.The radiochemical yield was obtained by measuring the initial activity and the activity lost during the synthesis and dialysis.For the first method, the results showed a loss of approximately 25% of the initial activity in the dialysate.With the second method, the results demonstrated a continuous increase in 223 Ra in the dialysate activity, up to a maximum of 43.6 ± 2.4% for the NPs radiolabeled with the second method.The final radiochemical yields were 34.1 ± 2.9% and 72.3 ± 3.0%, respectively [32].
Liposomes are polymeric NPs can be directly radiolabeled with radionuclides by an ionophore-mediated process.The radionuclide was first added to a vial containing a Ca − ionophore film, and the pH was adjusted to 7.4.Liposomes were added to the solution and incubated at 65 • C for 30 min.The addition of enediamine-N and Nl-tetraacetic acid (EDTA) in phosphate-buffered saline (PBS) quenched the loading, and then the mixture was kept in the rest phase for 10 min.Purification was accomplished using size-exclusion chromatography with a PD-10 column.The researchers found that temperature was a significant parameter for the high uptake of the 223 Ra into the liposomes.The rate of incorporation was <2% at 45 min, and it increased to 78 ± 6% in the final condition [33].
Nanomicelles are colloidal structures with a hydrophilic outer layer and a hydrophobic inner layer formed by amphiphilic monomers.Nanomicells are thermodynamically more stable than conventional micelles, and they are known for their excellent drug-delivery system.[35].
Iron-oxide NPs can be radiolabeled without with a chelator utilizing a heat-induced metal-ion-binding method, which avoids the use of a multi-step radiolabeled process by using a diagnostic radioisotope in the indirect approach [4].
Gholami et al. developed radiolabeled NPs with superparamagnetic iron oxide (SPIO) cores, commonly known as Feraheme ® (FH) NPs, in which radioisotopes bind directly to the surface of the SPIO core.The therapeutic radioisotopes used were 223 Ra, 177 Lu, 90 Y, and 64 Cu.The study showed that the FH NPs could be radiolabeled with high RCY and RCP using both diagnostic radioisotopes with long-range beta emitters and therapeutic radioisotopes with short-range alpha emitters [36].
Gawęda et al. radiolabeled barium-ferrite nanoparticles (BaFe NPs) with 223 Ra.Iron chloride and barium chloride were mixed in an 8:1 molar ratio (Fe 3+ :Ba 2+ ), and then 223 Ra chloride salt was added, followed by sodium hydroxide.The percentage of 223 Ra incorporated was calculated from the ratio of the radioactivity retained inside the nanoparticles to the radioactivity of the 223 Ra initially added, producing 61.3 ± 1.8% [37].
Suchánková et al. compared two different approaches to radiolabeling with hydroxyapatite NPs.The radionuclide was directly coupled with the surfaces of previously synthesized NPs in the first approach.After dispersing the NPs in a physiological saline solution, 223 Ra was added.The samples were mixed for 1 h at room temperature and then washed thrice with saline.The second approach was used to incorporate radionuclides into the structures of nanoparticles by adding Ca(NO 3 ) 2 to demineralized water.Both methods showed high RCY (≥94%), demonstrating that they are both feasible strategies for labeling, with the caveat that the daughter radionuclides were not determined [38].
Graphene quantum dots (GQDs) were chosen from among carbon-based nanomaterials for direct radiolabeling with 223 Ra.The radiolabeling process was simple; after the addition of EDC in a Britton-Robinson buffer in a GQD-FA solution, an aliquot of [ 223 Ra]RaCl 2 was added, and the mixture was heated under stirring at 90 • C for 1 h.To demonstrate the presence of radiolabeled NPs, X-ray spectroscopy (EDS), was performed after two and fourteen days of radiolabeling, associating the presence of 223 Ra with the formation of 211 Pb and 207 Pb as products of 223 Ra decay [39].
Piotrowska et al. radiolabeled nanozeolites with 223 Ra by exchanging the Na + with the 223 Ra 2+ cations in the zeolite structure.A 223 Ra(NO 3 ) 2 solution was added to the nanozeolite sample, which was then sonicated in an ultrasound bath for 15 min and gently shaken for 2 h.Next, the suspension was centrifuged at 13,000 rpm for 10 min and separated from the supernatant.The findings suggested a successful radiolabeling, with a high affinity and stability of 223 Ra [34].Mokhodoeva et al. radiolabeled superparamagnetic iron-oxide nanoparticles (SPIONs) with 223 Ra and reported an analysis of the 223 Ra uptake by the Fe 3 O 4 SPIONs.The results showed that the uptake speed of the radium was moderate, and 30-60 min were required for the radiolabeling with SPIONs, with yields of over 85%, ranging up to 99% [40].
Subsequently, the same research group studied the release of 223 Ra, 211 Bi, and 211 Pb recoils from radiolabeled NPs [45].The results showed the low release of all the isotopes from the NPs, depending on the method used to separate them (dialysis or centrifugation).With these experiments, the authors highlighted that false-positive results of radiolabeled NP stability can occur and, in particular, the recoiling progeny may induce artifacts.In conclusion, the authors suggested that in vitro stability tests should always be designed under real or very-similar-to-real conditions [45].

Discussion
The radiolabeling of 223 Ra with NPs using BFCs is a complicated process due to the properties of the radioisotope; this incorporation can be achieved by the direct radiolabeling method.Studies highlight strategies that allow direct radiolabeling; however, certain restrictions are associated.For example, Radon-219 ( 219 Rn) is a daughter isotope of 223 Ra, whose in vivo redistribution is unknown.Further investigation into the biodistribution of 223 Ra and its daughter isotopes is required for future clinical application.

Radiolabeling of NPs with Beta-Emitting Isotopes
Beta (β) emitters are the most commonly used radionuclides in therapy.These βemitting radioisotopes have the largest particle range (≤12 mm) and lowest linear energy transfer (LET) (0.1-1.0 keV µm −1 ).The β particles emit electrons that interact with atoms by passing through tumor tissues and losing their energy, leading to the release of ionized and excited atoms, as well as free radicals, which cause single-strand breaks in DNA and cell damage.However, this damage can also affect healthy tissue around tumor sites [46][47][48].The methods for radiolabeling NPs with alpha-emitting isotopes are summarized in Table 3.

Radiolabeling with Gold-198
The radionuclide Au-198 ( 198 AuCl 4 ) has a half-life of 2.7 d with cyclotron production and β and γ decay.The main advantage of the application of this isotope in nanomedicine is that the radiochemical compound is often the same element of the NP structure (AuNPs).

Direct Radiolabeling
Extensive studies of AuNPs have been carried out for a wide range of applications.Their characteristics, such as their easy synthesis process, the possibility of their functionalization, their inertness, and their peculiar optical properties, make them interesting candidates for both diagnostic and therapeutic purposes [114].Indeed, most studies used AuNPs to synthesize 198 AuNPs for radiation therapy (RT) with the goal of delivering a lethal dosage of radiation to tumor lesions while avoiding exposure to surrounding healthy tissues.The synthesis of 198 Au-labeled NPs differs significantly from that with other isotopes.Most investigations are based on the neutron irradiation of the gold foil, which produces radiolabeled NPs.The gold foil is irradiated by a neutron flux from a 198 Au reactor, and then dissolved in aqua regia, followed by the addition of HCl, after which it is allowed to evaporate the solvent.
This evaporation process is repeated three times, and then the specimen is left at room temperature for the crystallization of H 198 AuCl 4 •3H 2 O. Once the radioactive precursor is synthesized, the next step is conjugation with additional molecules to improve the specificity of the NPs.
Several molecules are used for this purpose: arabinoxylan (AX), a hemicellulosic material with a colon-specific uptake; gum arabic glycoprotein (GA), a plant extract approved by the FDA to target tumor cells; epigallocatechin-gallate (EGCg), which was shown to be beneficial in treating brain, prostate, cervical, and bladder cancers; mangiferin (MGF), a prostate-tumor-specific natural substance; cyclic arginine−glycine−aspartate peptide (RGD), known as a peptide that can specifically bind to integrin-αvβ3 expression in tumor angiogenesis; and, finally, human serum albumin (HSA) or apolipoprotein E (apoE).
Conjugation with these molecules often requires a reducing agent, such as N 2 H 4 or NaBH 4 , to reduce Au 3+ ions to Au 0 , stabilizing the isotope.
The characterization of NPs can be performed in different ways: by surface plasmon resonance (SPR), in which absorption at 526 nm is used to determine the shape and the particle size; by transmission-electron microscopy (TEM); by ultraviolet-visible spectrophotometry, in which an absorption band around 540 nm is characteristic of AuNPs; by high-resolution γ-ray spectrometry using a HPGe detector; or by nuclear magnetic resonance (NMR).
The particle size is frequently measured using dynamic light scattering (DLS), and a DLS analysis of AuNPs revealed the formation of very small nanoparticles (12-18 nm), while Zetasizer Nano ZS confirmed the polydispersity index (PDI) of 0.106 [35,[48][49][50][51][52][53][54].The surface charges of nanoparticles are an important factor in influencing their biological properties.The biodistribution of AuNPs is affected due to surface charge, such as in the kidneys, positively charged particles are used to accumulate, while a high accumulation of negative and non-charged particles was shown in the liver [15].Hirn et al.  radiolabeled 198 Au with 1.4-, 5-, 18-, 80-, and 200-nanometer negatively charged AuNPs and 2.8-nanometer positive charged AuNPs.The biodistribution of AuNP was measured by gamma spectrometry after 24 h of intravenous administration into rats.The results showed that the liver accumulation increased from 50% of 1.4-nanometer AuNPs to more than 99% of 200-nanometer NPs, while most other organs had small size-dependent accumulations of 18-200-nanometer AuNPs.However, the accumulation of AuNPs between 1.4 nm and 5 nm increased significantly with a decrease in the particle size, showing a linear relationship with the volumetric specific surface area [115,116].
The development of both nanobiological interfaces and their potential treatment requires an understanding of the in vivo effects of the surface chemistry of AuNPs.Wang et al. investigated the in vivo biodistribution, excretion, and toxicity of three different surfacecharged glutathione-protected gold nanoclusters (AuNCs), but with equal hydrodynamic sizes.In addition, the authors analyzed the potential application of these nanoclusters in radiation therapy and tumor uptake.The findings revealed that the surface charge significantly affected the pharmacokinetics, specifically in terms of their renal excretion and accumulation in the liver, testes, kidneys, and spleen.The peripheral blood system was temporarily affected by positively charged clusters, while negatively charged Au NCs had lesser excretion and higher tumor uptake, implying the potential of NC-based therapeutics [117].

Discussion
As discussed above, a research reactor is needed to synthesize 198 Au-labeled NPs.This approach allows radiolabeling with high radiochemical yields, but it can only be used during the synthesis of NPs.

Radiolabeling with Holmium-166
Holmium-166 ( 166 Ho) is a radioisotope with a half-life of 26.9 h; it is used in gamma scintigraphy by combining the emission of therapeutic β − particles with γ rays [118].
These features make 166 Ho an ideal isotope for theragnostic purposes.Furthermore, 166 Ho is a paramagnetic and electron-dense element that can be used for X-ray computed tomography (CT) and magnetic resonance imaging [119][120][121].

Direct Radiolabeling
Holmium-acetylacetonate microspheres (HoAcAcMS) were studied by Bult et al.Once synthetized, the samples were irradiated with a thermal neutron flux of 5 × 1012 cm −2 s −1 for 3 or 6 h, and the radioactive 166 Ho was left to decay closed in vials for one month.The degradation of the microspheres (the release of Ho 3+ ions from HoAcAcMS) was evaluated by suspending 100 µL of 166 HoAcAcMS in 2% Pluronic ® F68 aqueous solution and incubation in test tubes containing an isotonic phosphate buffer.The test tubes were placed in a continuously shaken water bath at 37 • C and centrifuged at different times (1 day, 4 days, and 1, 2, 4, 8, 12, and 24 weeks).The supernatant was analyzed after six months of incubation in the buffer and showed a release below 0.5%, demonstrating the stability of 166 HoAcAcMS.This was also confirmed in in vivo, by studies carried out in VX2tumor-bearing rabbits, where no release of holmium was observed, and the microspheres remained intact in the tumor tissue for one month [111].
Electrospinning is a simple and low-cost technology for producing nanoparticleembedded fibers with homogeneously dispersed NPs.Munaweera et al. incorporated non-radioactive holmium-165 ( 165 Ho) iron-garnet nanoparticles (HoIGNPs) into a bandage by using the electrospinning method for neutron irradiation.The polymer fibers were stable following neutron activation, with no leakage of radioactive material [112].
The radiolabeling process can also be performed after synthesizing nanoparticles without using a chelator.Vimalnath et al. radiolabeled agglomerated IONPs by mixing them with a 166 HoCl 3 solution (obtained by radiative neutron capture) and stirring the mixture at room temperature for 60 min.The 166 HoIONPs were separated from the supernatant, after which the unlabeled residue was purified by washing with a sterile solution.The findings demonstrated that a minimum amount of IONPs (5 mg of particles in 1 mL of reaction volume) achieved a radiolabeling yield of more than 95%.The yield was excessively low at pH levels below 5, whereas no significant changes were detected at pH levels between 5 and 10.As a result, the optimal pH was determined to be 7-8, which is advantageous, since it is close to the physiological pH [113].

Discussion
We reported the neutron irradiations and the electrospinning approach to radiolabel NPs with 166 Ho.The neutron-irradiation method is effective but time-consuming, whereas electrospinning is simple and cost-efficient.However, both techniques led to a high radiochemical yield with a non-significant release of free 166 Ho over time.

Radiolabeling with Yttrium-90
Yttrium-90 ( 90 Y) is a β-emitter with a half-life of 64.1 h and a high average level of energy (2.27 MeV), making it ideal for radionuclide therapy for large tumors, since it can damage tumor cells up to a depth of 11 mm in soft tissue [122].Nevertheless, significant damage can also occur to neighbor normal tissues.

Direct Radiolabeling
Magnetic nanoparticles (MNPs) are found to be suitable for direct radiolabeling with 90 Y. Radović et al. synthesized Fe 3 O 4 -MNPs and Fe 3 O 4 functionalized polyethylene glycol 600 diacid (PEG600)-MNPs to compare their efficacy as radioactive vectors.The radiolabeling was achieved by mixing the NPs solution with 90 YCl 3 and stirring the reaction mixture for 1 h at room temperature.The results showed high radiolabeling yields for both MNPs: 97% for the naked Fe 3 O 4 and 99% for the Fe 3 O 4 -PEG600 [55].
Ognjanovíc et al. used 90 Y and 177 Lu with IONP-coated citric acid (CA), poly(acrylic acid) (PAA), and poly(ethylene glycol) (PEG).The radiolabeling process was based on incubating the radionuclide with the IONPs for 30 min or 60 min.The iTLC confirmed that the radiolabeling yields were greater than 99% for every sample except for the 177 LuPEG-IONP, regardless to the incubation time [56].
In another work, Radović et al. designed 90 Y-labeled imidodiphosphate-and inositolhexaphosphate-coated magnetic nanoparticles.Their iTLC analysis confirmed that the radiolabeling yield from the coated-MNPs was greater than 98% [57].
Soni et al. radiolabeled YPO 4 :Er 3+ −Yb 3+ NPs, rare earth (RE) NPs, with 90 YCl 3 or 177 LuCl 3 , without the use of any BFC.The binding equilibrium for both radioisotopes was achieved within 30 min of incubation at pH 6.5; after the purification, both formulations achieved a radiochemical purity of more than 95% [58].

Indirect Radiolabeling
Diethylenetriaminepentaacetic acid (DTPA) and DOTA are primarily used, as BFCs, for radiolabeling NPs with 90 Y.
Li et al. used DTPA for the radiolabeling of liposomes.The liposomes were conjugated with DTPA in a chloroform solution.The chloroform was then removed by evaporation at 60 • C, water was added to obtain an aqueous solution, and 90 YCl 3 was added at room temperature for 30 min.The RCY was evaluated using a nanosep filter in a microfuge at 4000 rpm, and the results showed that more than 95% of the 90 Y bound to the liposomes [59].
Buckway et al. used a N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer to synthesize polymeric NPs and radiolabeled them with DOTA.The HPMA-DOTA copolymerization was achieved using reversible addition-fragmentation-chain transfer (RAFT), after which 90 YCl 3 was added, and the solution was incubated for 1 h at 50 • C under nitrogen.EDTA was added to the solution after it reached room temperature to eliminate any free or loosely bound 90 Y 3+ ions.The results showed that DOTA may be a stable chelator for this radioisotope [60].

Discussion
As discussed above, the negative surface charging of nanoparticles is required for direct radiolabeling with positively charged 90 Y 3+ .If the native NPs do not have a negative charge, they can be coated with multiple molecules without affecting their chemicalphysical properties.Furthermore, different BFCs can effectively be used for indirect radiolabeling.

Radiolabeling with Lutetium-177
Lutetium-177 ( 177 Lu) has a half-life of 6.7 days; it is considered ideal for the theragnostic emission of γ photons and β − particles, which penetrate only 2 mm into tissues and, thus, spare normal neighboring tissues or cells from irradiation.In addition, 177 Lu has been used in radio-synovectomy and hepatocellular carcinoma therapy, and it is currently used for several new treatments for cancers, including neuro-endocrine tumors [123] and prostate cancer [124].

Direct Radiolabeling
By modifying the surfaces of MNPs, to design potential theragnostic agents, these can be radiolabeled as chelator-free with high radiochemical purity and stability.Salvanou et al. radiolabeled alginic coated (MA) IONPs with 177 Lu ( 177 Lu-MA) and also stabilized them with a layer of polyethylene glycol (PEG) ( 177 Lu-MA-PEG).The iTLC-SG analysis confirmed values of 95.2 ± 1.3% RCY for the 177 Lu-MA and 93.6 ± 1.0% for the 177 Lu-MA-PEG [61].
Poly-L-lysine, proline, and tryptophan can also be used for MNP coating.Mirković et al. fabricated poly-L-lysine coated MNPs (PLL-MNPs) and modified IONPs with proline (Pro-MF) and tryptophan (Trp-MF).These NPs were radiolabeled with two therapeutic radioisotopes, 177 Lu and 131 I.The radiolabeling yield was quantified by iTLC, which showed that the highest radiolabeling yield was for the PLL-MNPs (99% for both formulations), while the Pro-MF and Trp-MF yielded 59.5% and 45.6%, respectively, at room temperature, and 69.2%, and 70.3% at 80 • C [62].
Joshi et al. recently published an interesting report on the radiolabeling of NaGdF 4 /Ho-Yb@m-SiO 2 nanoparticles (UCNPs) with 177 Lu, obtaining 98.4 ± 0.4% RCY, and studied their biodistribution in rats, showing no uptake of free 177 Lu in bones up to 168 h [64].
Chakraborty et al. radiolabeled hydroxyapatite NPs with 177 Lu.Their results showed a high radiolabeling yield (99.1 ± 0.2%), and no radioactivity of the free radioisotope was detected [66].

Indirect Radiolabeling
Among the BFCs, 177 Lu can form a stable coordination complex with DOTA or with its derivates.The radiolabeling process can be performed either by labeling NPs with 177 Lu-BFC or by labeling BFC-conjugated NPs.
For the first strategy, the radiolabeling of DOTA with 177 Lu required an incubation time at a high temperature (80-90 • C) and a purification step to remove the unbound 177 Lu.Next, conjugation with AuNPs was performed in mild conditions under stirring for a few minutes.The results showed that DOTA can efficiently bind to AuNPs.Indeed, the radiochemical purity in all the published papers was >90% [84][85][86][87][88][89][90].
The second strategy was chosen by several other authors, who chose a very similar methodology.
The DOTA was not only used with AuNPs but also with several other types of NP, such as PLGA-NPs.To conjugate the chelator, lyophilized PLGA-NPs were dissolved in water, DMF, DIPEA solution, and carboxylate-activating agent (HATU).The DOTA was added subsequently, and the mixture was incubated under slow stirring for 2 h at room temperature.Next, a solution of 177 LuCl 3 was added in an acetate buffer (pH 7) to the DOTA-PLGA-NP solution and was then heated at 37 • C for 3 h, producing radiolabeled NPs with high radiochemical purity [70,71].
In addition, DOTA can be conjugated with NPs during the synthesis of particles, as demonstrated by Wang et al. when they radiolabeled DOTA-tri arginine-lipid with 177 Lu [75].
Reconstituted high-density lipoproteins (rHDLs) are promising platforms for developing theragnostic systems due to their ability to transport and release drugs and imaging agents through the scavenger receptor (type B1 (SR-B1)) to tumor-targeting sites.Aranda-Lara et al. studied 177 Lu-radiolabeled rHDLs, using DOTA as a chelator.The radiochemical yield was 85% before the purification (performed by centrifugation) and 99% after the purification.Moreover, these NPs showed high stability, with a radiochemical purity >95% after 24 h of incubation at 37 • C in saline and human serum [77].
Zhang et al. fabricated alpha-melanocyte-stimulating hormone (αMSH)-surfaceengineered PEG-coated silica NPs and radiolabeled them with 177 Lu by using DOTA as a chelator to target the melanocortin-1 receptor (MC1-R) in melanoma-tumor cells.The results showed a radiochemical yield of more than 95% [79].
Shultz et al. investigated the theragnostic effect of radiolabeled 177 Lu and 177 Lu-DOTA with functionalized metallofullerene (Gd 3 NC80) NPs ( 177 Lu-Gd 3 NC80 and 177 Lu-DOTAf-Gd 3 NC80) in brachytherapy.The results showed radiopurity of 80%, while the in vivo stability study revealed some 177 Lu-DOTA degradation or cleavage from the f-Gd 3 NC80 surface.Despite this level of in vivo degradation, this investigation demonstrated effective brachytherapy by comparing 177 Lu-labeled f-Gd 3 NC80 to f-Gd 3 NC80 alone [80].D'Huyvetter et al. designed 177 Lu-nanobodies by conjugating DOTA and DTPA chelators to target HER2-positive breast cancer.Both formulations showed radiochemical purity of more than 98% in the iTLC analysis [81].
In addition, DTPA was used for liposomes.The conjugation between the chelator and the NPs was performed during the synthesis of the liposomes, which involved mixing the lipids in chloroform-methanol and dimyristoylphosphoethanolamine-DTPA (DMPE-DTPA) in two different molar ratios to achieve two formulations, with "high" DTPA loading and with "low" DTPA loading.The radiolabeling was performed by simply adding a solution of 177 LuCl 3 to the liposomal solutions and incubating the solutions at room temperature for 30 min.The RCY was higher than 80%, but a high amount of NPs was necessary.This was due to the higher amount of carrier-lutetium in this radionuclide, which resulted in a higher mass of lutetium per radioactivity, which led to achieving a lower SA [82].
Ge et al. reported that Fe 3 O 4 NPs can be labeled with 177 Lu using DP-PEG as a chelating agent.They also labeled these NPs with 99m Tc for SPECT and with 68 Ga for PET imaging.The RCY was approximately 50%, but the labeling was stable and did not modify the physical properties of the NPS [67].

Radiolabeling by Encapsulation
The encapsulation of radioisotopes in nanoparticles may prevent biological degradation, and, as a result, lower concentrations may be sufficient to achieve the desired effects.Chakravarty et al. developed a human serum albumin (HSA)-mediated biomineralization process for the synthesis of intrinsically radiolabeled 177 Lu 2 O 3 nanoparticles entrapped in a protein scaffold ( 177 Lu 2 O 3 -HSA nanocomposite).The radiochemical purity of the 177 Lu 2 O 3 −HSA nanocomposite was determined by radio-thin-layer chromatography (radio-TLC), and a purity rate of more than 98% was achieved [65].
Arora et al. fabricated 177 Lu-DOTA-TATE-loaded PLGA nanoparticles (NPs).The results showed that the encapsulation efficiency (EE) was between 77.3 ± 4.9% and 58.4 ± 5.3%, depending on the experimental condition.The authors showed that PLGA-NPs also have a slower release rate for the isotope [83].

Discussion
The direct method of radiolabeling NPs with 177 Lu requires high temperatures and may disrupt the NPs; on the other hand, the indirect method may take a long time under acidic pH [124].To overcome these limitations, the radiolabeling of NPs can be performed with 177 Lu-DTPA.The encapsulation approach for radiolabeling is preferable because of its excellent yield and reproducibility under mild and physiological conditions.

Radiolabeling with Iodine-131
It is known that 131 I, which has a radioactive decay half-life of 8.02 days, is the most commonly used β-particle-emitting radioisotope in nuclear medicine for treating thyroid cancer, neuroblastoma, and pheochromocytoma [125].
The emission of γ-rays makes it a potential radioisotope for molecular imaging [3].The radiolabeling of NPs with iodine radioisotopes is based on halogen-nucleophilic exchange, which can be conducted directly with an oxidizing agent via the direct radioiodination of NPs, or indirectly, by employing prosthetic groups.

Direct Radiolabeling
Chen et al. radiolabeled palladium (Pd) nanosheets with 131 I by using the direct method without using an oxidizing agent.The results showed a labeling efficiency of more than 98% [110].
Among the oxidizing agents, the 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril (Iodogen) method and the N-chlorobenzenesulfonamide (chloramine-T) method are the frequently most used.The first is a less aggressive process based on the dissolution of Iodogen in an organic solvent and evaporated in the presence of a tube, remaining bound to the wall.The second is a strong oxidant that leads to radioiodination in a few seconds, but a reducing agent is required to stop the reaction.To control the oxidizing activity without affecting the biomolecules present in the solution, polystyrene beads are used to fix the activity; thus, the oxidizing reaction is stopped when the beads are removed from the solution [126].
The Iodogen method allows the radiolabeling of several types of NPs.˙Ince et al. synthesized the glucuronide derivative of thymoquinone (TQG) enzymatically, conjugated it with a synthesized MNP, and then radio-iodinated the mixture with 131 I.The findings suggested that the nanoparticles were radio-iodinated with 131 I with a yield of over 95% [92].
Chen et al. developed albumin-based gadolinium-oxide NPs (GdNPs) loaded with doxorubicin (DOX) and conjugated with bone-seeking alendronate (ALN) to treat malignant bone destruction.The abundance of tyrosine residues in the albumin molecule resulted in a high radiolabeling efficiency [107].
Using the chloramine-T method, Zhao et al. radiolabeled 131 I to the surface of polydopamine (PDA)-coated SWCNT modified with PEG to increase blood-circulation half-life (SWCNT@PDA-PEG).The results showed a radiolabeling yield of approximately 90% [97].Several other authors used the chloramine-T method [99,100,102,103], with RCY values varying from 55% to 90%, suggesting the need for the further purification of radiolabeled NPs from unbound iodine.As an alternative to chloramine-T, Liu et al. used NaClO 4 as an oxidizing agent to oxidize iodine from I − to I + .They labeled hollow copper-sulfide NPs with paclitaxel, thus combining phototermal, chemo-and radio-therapies [109].

Indirect Radiolabeling
The Bolton-Hunter reagent, the N-hydroxysuccinimide ester of iodinated p-hydroxyphenyl propionic acid, is the only BFC utilized for iodine radioisotopes.It is used to iodinate molecules that do not have tyrosine residues, allowing them to be radiolabeled with NPs.
Its precursor, 3-(4-hydroxyphenyl)propionic acid-OSu (HPAO), was also used to radiolabel NPs, particularly for the radiolabeling of polyethyleneimine-entrapped gold nanoparticles (Au PENPs) and PAMAM dendrimers.For the former, polyethyleneimine (PEI) was selected as a template for the further modification with PEG, chlorotoxin, a gliomaspecific peptide (CTX), and HPAO, which were then used to entrap gold nanoparticles (Au NPs).Conjugation with HPAO was achieved by adding HPAO to the template after the modification with PEG and CTX, and stirring overnight.After trapping the AuNPs in the modified PEI,PENP-CTX was obtained.The radiolabeling process was performed by, first, mixing the Na 131 I solution with chloramine-T and PENPs-CTX with continuous stirring.The final mixture was incubated for 30 min at 37 • C and was then purified with a PD-10 desalting column.The radiochemical purity (determined by ITLC) was measured at different times to evaluate the radio-stability, showing a radiochemical purity of over 99%, remaining above 90% after 24 h in PBS at room temperature and in FBS at 37 • C [97,99].
Song et al. synthesized 131 I-labeled dendrimers modified with the LyP-1 peptide as a multifunctional platform for single-photon emission computed tomography (SPECT) imaging and radionuclide therapy for metastatic cancer.The iTLC analysis confirmed a radiochemical purity of more than 99% and radiochemical stability of more than 90% [96].
Finally, Ji et al. radiolabeled albumin nanospheres loaded with doxorubicin using a new chelating agent, namely N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP).Firstly, iodine was bound to a monoclonal antibody (anti-AFPMc), and then the radiolabeled antibody was linked to the NP via the SPDP cross-linker.It was not possible to use the resulting construct to perform chemo-radiotherapy for liver cancer.The results obtained from Balb/c mice with HepG2 xenografts showed improved tumor reduction compared with mice treated with doxorubicine alone [100].

Radiolabeling by Encapsulation
The chloramine-T method was used for labeling BSA encapsulated into the cores of liposomes.The encapsulation of the radionuclide was possible due to the capacity of the oxidized 131 I 2 to pass through the phospholipid bilayer of the liposome and react with the tyrosine residue present on the BSA.The oxidation was carried out by mixing the liposomes with K 131 I and chloramine-T.The solution was vortexed for 10 min, and then purified using a Sephadex G-100 column.The radiolabeling yield, measured by a gamma counter, was over 90% [106].
Sakr et al. synthesized polyethylene-glycol-capped silver nanoparticles doped with I-131 radionuclide ( 131 I-doped Ag-PEG NPs).The radiolabeling yields and the encapsulation efficiency of the Ag-PEG NPs were studied at room temperature for up to 8 h.The maximum radiolabeling yield (98 ± 1%) was obtained after 3 h of stirring, and there was no 131 I-leakage from the Ag-PEG NPs until 6 h post-synthesis [108].

Discussion
The radiolabeling of NPs with 131 I typically involves the electrophilic aromatic substitution of phenolic or trialkylstannylated substrates using an oxidant agent, such as chloramine-T or Iodogen [127].With these methods, high radiochemical yields are obtained.The main limitation of iodine-labeled NPs is deiodination, with the undesired accumulation of radionuclides in normal tissues, such as the thyroid and stomach.Therefore, the fabrication of stable and multifunctional nanocarriers for the efficient delivery of radioactive iodine to targets is required [127].

General Conclusions
There has been a significant increase in interest in integrated research at the interface of nuclear medicine and nanotechnology over the last two decades [126,128].This developing convergent science can potentially overcome the limitations of current radionuclide therapy.This review highlights the radiolabeling of NPs with Auger's electron-emitting isotopes, alpha-emitting isotopes, and beta-emitting isotopes by direct, indirect, and encapsulation methods.Nanostructures with linked Auger's electron emitters have a substantial advantage over traditionally labeled biomolecules, delivering significantly more radioactivity to targeted tissues.This is important for Auger's electron therapy since it needs considerably higher radionuclide activity to achieve a therapeutic effect.Therapeutic β-emitting radioisotopes are found in various organic, inorganic, and hybrid forms of nanomaterials.Radiolabeling is typically accomplished through the use of a bifunctional chelator or through the formation of covalent bonds.The easy availability of β-emitters and successful results may enable the development of various nanoplatforms for effective theragnostic research.The α-particle-incorporated nanomaterials have received less attention because of their higher production costs and lower availability.The encapsulation of radionuclides within nanostructures was preferred for generating stable therapeutic agents because commonly employed chelating agents or labeling processes typically cannot sequester the α-emitter and its decay products.
Nanoparticles can be radiolabeled with radioisotopes by multiple methods to allow multimodal therapy.The specific therapeutic application of NPs is based on the most appropriate choice of nanomaterials and isotopes, and the most suitable radiolabeling method.Indeed, all these factors influence the results and the possible application of the method to humans.
As shown in this review, different methods for radiolabeling NPs have been proposed.Therefore, it is essential to standardize these processes and develop reproducible protocols to apply them for clinical purposes.

Biomolecules 2022 , 33 Figure 1 .
Figure 1.Publications on nanoparticles and on radiolabeled nanoparticles for diagnostic SPECT/PET use from 2012 to 2022.

Figure 1 .
Figure 1.Publications on nanoparticles and on radiolabeled nanoparticles for diagnostic SPECT/PET use from 2012 to 2022.

Table 1 .
Particles and labeling methods with Auger's electron emitter.

Table 2 .
Particles and labeling methods with α emitters.
Souza et al. synthesized nanomicelles of [ 223 Ra]RaCl 2 co-loaded with [ 198 Au]AuNPs.Non-radioactive AuNPs were synthesized and irradiated for 12 h in an Argonauta reactor to produce [ 198 Au]-AuNPs.Subsequently, the [ 223 Ra]RaCl 2 nanomicelles were synthesized, and a Pluronic F127 copolymer was dispersed while stirring.Next, [ 198 Au]AuNPs were added to the solution and ultrasonicated for more than 2 min in an ice bath at 10 • C. The in vitro citotoxicity studies revealed a substantial increase in tumor-cell death when the treatments with both [ 223 Ra]RaCl 2 and [ 198 Au]Au-NPs were combined in the same formulation, compared to the [ 223 Ra]RaCl 2 or [ 198 Au]Au-NPs alone