Nanoparticles labeled with gamma-emitting radioisotopes: an attractive approach for in vivo tracking using SPECT imaging

Abstract

Providing accurate molecular imaging of the body and biological process is critical for diagnosing disease and personalizing treatment with the minimum side effects. Recently, diagnostic radiopharmaceuticals have gained more attention in precise molecular imaging due to their high sensitivity and appropriate tissue penetration depth. The fate of these radiopharmaceuticals throughout the body can be traced using nuclear imaging systems, including single-photon emission computed tomography (SPECT) and positron emission tomography (PET) modalities. In this regard, nanoparticles are attractive platforms for delivering radionuclides into targets because they can directly interfere with the cell membranes and subcellular organelles. Moreover, applying radiolabeled nanomaterials can decrease their toxicity concerns because radiopharmaceuticals are usually administrated at low doses. Therefore, incorporating gamma-emitting radionuclides into nanomaterials can provide imaging probes with valuable additional properties compared to the other carriers. Herein, we aim to review (1) the gamma-emitting radionuclides used for labeling different nanomaterials, (2) the approaches and conditions adopted for their radiolabeling, and (3) their application. This study can help researchers to compare different radiolabeling methods in terms of stability and efficiency and choose the best way for each nanosystem.

Graphical Abstract

Introduction

Diagnostic radionuclides and radiopharmaceuticals

Radioactive nuclides (radionuclides) release such particles as a positron (β⁺), negatrons (β⁻), alpha (α), electrons (e), or γ-rays to balance their nuclear forces. The application of radionuclides as diagnostic, therapeutic, or theranostic (diagnostic and therapeutic) determines in terms of their decay products. The gamma- and positron-emitting radionuclides are used for diagnostic aims. The radionuclides with highly entrapped nuclear energy undergo the “isomeric transition” and subsequently release their energy by emission of a single γ photon. The neutron-deficient radionuclides emit an unstable positron particle which converts into positronium in collision with a surrounding electron. This phenomenon is called “annihilation,” and the release of two gamma rays in opposite directions with specific energy (0.511 MeV) is its consequence. Diagnostic radiopharmaceuticals, as a well-known class of drugs, are composed of a diagnostic radionuclide (metallic/non-metallic) that is bound to a carrier. Various carriers, including small organic molecules, peptides, monoclonal antibodies, cells, and nanoparticles, could be labeled with these diagnostic radionuclides using different strategies [1].

Nuclear imaging modalities

Two nuclear modalities are applied to obtain high-quality images from deep tissues/organs to assess their functions. Single-photon emission computed tomography (SPECT) is an imaging system designed for gamma-emitting radionuclides. The positron-emitting radionuclides can be detected by positron emission tomography (PET) imaging scanners. The most widely used radionuclides in SPECT and PET are technetium-99 m (^99mTc) and fluorine-18 (¹⁸F), respectively [2, 3].

Out of the imaging techniques, nuclear imaging systems are well-known owing to their high prediction accuracy, sensitivity in the picomolar range and no limit in the depth of γ-ray penetrations. In SPECT imaging, the emitted gamma rays can be detected by a gamma camera which is rotated around the subject or patient and capture the gamma rays in three dimensions. Using the narrow collimators, the radiations emitted from body patient converge into a parallel beam and lead to SPECT images with better resolution. In PET systems without a collimator, the resolution of images are intrinsically higher than SPECT images. It is due to the ability to detect two gamma photons — in coincidence — created by the annihilation of a positron. Indeed, the circular full ring scanner in PET imaging system make it possible. Moreover, the sensitivity of PET is greater than SPECT owing to the absence of collimation in the former that enhance signal quantification. Nevertheless, SPECT imaging is less pricey and more available compared to PET imaging. In sum, much better image quality and lower cost are the main advantages of PET and SPECT scans, respectively.

Accordingly, radiolabeling of nanomaterial can provide an opportunity to quantifiably track them through the body using their minimum amounts. A variety of diagnostic radionuclides have been used for labeling nanoparticles as a gamma camera or SPECT imaging agents (technetium-99 m, indium-111, iodine-123/125, gold-198/199, iodine-131, etc.) and PET imaging agents (flourine-18, zirconium-89, copper-64, gallium-68, manganese-52, etc.) [4].

Radiolabeled nanoparticles

Numerous approaches have been adopted for the attachment of radionuclides to nanostructures. The type of radionuclide (metallic/non-metallic) or nanoplatform (organic/non-organic), radiolabeling conditions, and availability of facilities are influential parameters in their effective radiolabeling. Ideally, the labeling of nanoparticles should be done in a such easy way that large amounts of radionuclides firmly bind to the minimum amounts of nanoparticles without making significant changes in their structural integrity [5].

A combination of radionuclides with nanoparticles can be performed directly — into their structures — or mediated by some chemical compounds. In direct radiolabeling method, the radionuclides (metallic or non-metallic) are attached to a part of the nanoparticle structure (inside or outside) without any intermediate molecule. For instance, the radiometal ¹⁹⁸Au can be embedded into the crystal structures of Au nanoparticles, or such radiohalogens as ¹⁸F or ¹³¹I can be covalently bound on the tyrosine residue of proteins existing at the surface of EVs via electrostatic substitution. For metallic radionuclides, such as ⁶⁴Cu and ^99mTc, the radiolabeling process is frequently performed with the aid of bifunctional chelating agents (BFCA) conjugated to the nanostructures, though. In these cases, more stable metal complexes can be achieved. Indirect labeling of nanoparticles with non-metallic radionuclides is also applicable via conjugation of prosthetic groups to the thiol-, amine-, or carboxylate-functionalized nanoparticles. Indeed, BFCAs are chemical compounds comprising a metal coordination site and a reactive functional group that enables it to be bound to the carriers like nanoparticles. Unlike BFCA, the prosthetic groups are well-known agents for the covalent attachment of non-metallic radionuclides like radiohalogens to the nanoparticles. Of note is that the radiolabeled or non-radiolabeled forms of these BFCAs or prosthetic groups can be covalently conjugated to the nanostructures or incorporated into their structures via van der Waals or electrostatic interactions. The BFCA or prosthetic groups can be also radiolabeled before or after their attachment to their binding site on the structure of nanoparticles. The use of [^99mTc(CO)₃(H₂O)₃]⁺ precursor for radiolabeling of nanoparticles containing amino acid fragments (cysteine, histidine) or tridentate BFCA is an example of the pre-labeling procedures. In this radiolabeling method, the organometallic complex is firstly prepared using the Isolink® kit formulation (Mallinckrodt) and then the tridentate BFCA (like His-tag or nitrilotriacetic acid (NTA)) is substituted with the three water ligands.

In some cases, especially vesicular or porous nanomaterials, the previously prepared radio-complexes as radiolabeled ionophores or organic ligands transport the radionuclides into their hollow spaces. An ionophore is a lipophilic ligand that could reversibly bind to a radiometal ion. Then, this radio-complex could cross through the lipid membranes, and release its radiometal inside the cavity, and deliver it to the intravesicular metal chelating proteins or encapsulated drugs or chelating agents. Thus, ionophores could be applied for radiolabeling of vesicle-based nanoparticles. The latter agents are used in the “remote loading” method. In this radiolabeling mechanism, like ionophore-based labeling, the previously radiolabeled lipophilic organic ligands (e.g., ^99mTc-Hexamethylpropyleneamine oxime; HMPAO or ¹⁸⁶Re-N,N-bis(2-mercaptoethyl)-N0,N0-diethyl-ethylenediamine; BMEDA) can be remotely loaded inside the vesicular nanomaterials. Unlike the ionophore-assisted technique, the radiometal complex is adequately stable to remain intact inside the vesicle in remote loading. The point of this method is that the organic ligands should have functional groups with the ability to get charged or hydrophilic in the aqueous media of the vesicle in order for entrapment inside the cavity. Different radiolabeling methods are shown in Fig. 1.

Fig. 1

Radiolabeling methods used for vesicular nanoparticles (left): a surface radiolabeling using a chelator, b surface radiolabeling without using a chelator (direct labeling), c radiolabeling using a chelator embedded into the lipid bilayer, d direct attachment of radionuclide into the lipid bilayer, e transporting radionuclide across the bilayer assisted by ionophore and then radionuclide-complex formation via the encapsulated chelating agent, f transporting radionuclides using a lipophilic ligand which can passively diffuse into the bilayer and then entrap into the cavity (remote loading method), g transporting radionuclides across the bilayer assisted by a ionophore, and then radionuclide-complex formation via the encapsulated drug being of the anchoring groups; Radiolabeling methods used for non-vesicular nanoparticles (right): h surface radiolabeling using a chelator, i direct surface radiolabeling (chemical adsorption), j direct entrapment of radionuclide into a structure of nanoparticle (hot-plus-cold labeling method), and k attachment of radionuclide-chelator complex to the core structure of the nanoparticle

The chemical structures of some of the radiolabeling intermediates with the corresponding radionuclides are depicted in Fig. 2 [4]. In this review, we try to introduce the various radiolabeling methods used for each nanoparticle and describe the details of their labeling process and the features of radiolabeling, including radiolabeling efficiency or radiolabeling yield (RY), radiochemical stability (RCS), and specific activity (SA). Moreover, at the tables related to each nanoparticle, the different radiolabeling methods have been distinguished by the specific colors: direct labeling method (gray), chelator-based method (yellow), remote-loading (blue), and ionophore-assisted labeling (pink). According to a search of the Web of Science using the relevant keywords, there are many types of research on radiolabeled nanoparticles. Figure 3 provides an overview of the number of studies conducted on radiolabeling of each category of nanoparticles so that the larger ellipses indicate more studies for those structures.

Fig. 2

The radiolabeling-intermediates used for labeling nanomaterial with their corresponding radionuclides

Fig. 3

An overview of the number of studies conducted on radiolabeling of each category of nanoparticles: DDM, dendrimer; GNP, gold nanoparticle; MNP, magnetic nanoparticle; PM, polymeric micelle; GBNP, grapheme-based nanoparticle; PNP, protein-based nanoparticle; SNP, silica nanoparticle; QD, quantum dot

Radiolabeling process outcomes

The aim of various radiolabeling approaches is to efficiently label low amounts of nanoparticles with a high degree of activity and the lowest radionuclide leakage after their administration. In this context, radiolabeling efficiency or radiolabeling yield (RY) is the ratio of activity in the final product to the starting activity. The effective radiolabeling method includes a mechanism of loading of the largest amounts of radionuclides into nanoparticles’ structure with the lowest personnel radiation. Radiochemical stability (RCS) is another parameter that shows the strength of the radionuclide bonds in the complex. This parameter determines the percentage of radionuclide leakage from the radiolabeled agents over time [4]. Using the more stable radiocomplexes, the lower radioactivity accumulation can be observed in non-target tissues.

Moreover, specific activity (SA) should be considered for each radiolabeling approach, and it is the measured activity per unit mass of radiolabeled compound. The higher specific activity of radiolabeled nanoparticles is obtained in condition that the lower amounts of nanoparticle can be labeled with a high concentration of radioactivity. The radiolabeling approaches which can render higher specific activity for exposure problems following the administration of these radiolabeled nanoparticles.

A radiolabeling approach will ideally work when the higher amounts radionuclides (higher RY%) can be load into the minimum possible mass of nanoparticles (higher SA) with high stability (higher RCS%). Using an ideal radiolabeled nanoparticle with high RY%, SA, and RCS%, the quality of SPECT or PET images — based on the type of radionuclide used — will be improved so that we can understand the exact biodistribution behavior of the non-targeted radiolabeled nanoparticle. In the case of application of the targeted nanoparticles radiolabeled with gamma-emitting radionuclides, the specific delivery and accumulation of radioactivity will be undertaken and consequently high quality images can be obtained from the target tissues. In other words, the better radiolabeling outcomes make the nanoparticles more useful to detect targets as precisely as possible.

Radiolabeled organic nanomaterial for diagnostic purposes

Organic nanoparticles are nano-scaled particles constructed by organic compounds (primarily lipids or polymers) in different forms. These nanomaterials have gained significant attention in the pharmaceutical field. Liposomes, albumin, dendrimer, and polymeric nanoparticles are examples of organic nanoparticles extensively used as drug delivery systems, so some of them have been approved for commercial uses [6, 7].

When tumor size increases, the tumor needs a more independent blood supply leading to angiogenesis. Also, with increasing tumor size, the blood vasculature tends to be compressed and decrease the blood supply generating a hypoxic state in the tumor. This causes an enhanced permeability and retention (EPR) effect which could increase the vascular permeability in the tumor or inflammation site and nanoparticles leverage from EPR to accumulate in these lesions. Radiolabeling of nanomaterials allowed researchers to trace these systems and determine their biodistribution in vivo. In this regard, nuclear imaging modalities such as SPECT and PET provide an opportunity to monitor their pharmacokinetics and biodistribution. Due to the long biological half-lives of nano-based drug delivery systems, it is important to select radionuclides with proportional half-lives used for diagnostic or therapeutic purposes. For example, in SPECT imaging, γ-emitting radionuclides, such as ^99mTc (t_1/2 = 6 h) and ¹¹¹In (t_1/2 = 2.8 days), are preferred, and for PET imaging, ⁶⁴Cu (t_1/2 = 12.7 h) and ⁸⁹Zr (t_1/2 = 3.3 days) are favorable positron-emitting radionuclides [8].

Liposomes

Liposomes as spherical vesicles organized by assembling lipid bilayers have been widely studied for diagnostic and therapeutic applications, especially in anticancer drug delivery [4]. It is likely due to their substantial effects on the biodistribution and pharmacokinetics of the incorporated drug. The liposome pharmacokinetics depends on their characteristics such as size, surface charge, and route of administration as well [9].

The biodistribution of liposomal formulations could be determined by radiolabeling of the liposomes with diagnostic radionuclides [10]. Radiolabeled liposomes served as diagnostic tools for the visualization of tumors, inflammatory, infections, cardiovascular diseases, and therapeutic and theranostic agents [11]. The liposomes can be located in the inflamed tissues through passive or active targeting, so gamma-labeled liposomes have been used for imaging inflammatory or infectious lesions. For instance, ^99mTc-PEG-liposome has been applied to take images from patients with soft-tissue infection, septic arthritis, and autoimmune polyarthritis [12]. Furthermore, liposomes could be encapsulated with pharmaceuticals that have already been radiolabeled as radiopharmaceuticals [13].

Vescan, as the first radiolabeled liposome, was loaded with ¹¹¹In and used as an imaging agent. To label liposomes with ¹¹¹In, the combination of citric acid buffer, liposomes, and ¹¹¹InCl₃ was heated at 80 °C for 30 min. More than 80% of ¹¹¹In was loaded into the liposomes. Then, sodium edetate was used to chelate an excess of ¹¹¹In [14].

In another study, a liposome labeled with positron-emitting radionuclide ⁸⁹Zr was studied to evaluate its in vivo pharmacokinetic. The ⁸⁹Zr-labeled liposome encapsulated with deferoxamine (DFO) was traced in the KB tumor xenograft-bearing CD1 nude mice until 48 h post-injection. This radiolabeled nanosystem had no release of radioactivity in PBS (phosphate-buffered saline) and serum. PET imaging of ⁸⁹Zr-liposome exhibited that the radioactivity gradually washed out from the liver and kidney and deposited in the bone tissues. The high hepatic catabolism of ⁸⁹Zr-DFO-liposomes led to the release of ⁸⁹Zr into the circulation, which tends to accumulate in the bone tissue [15]. The fate of liposomes has also been assessed using ^99mTc- and ¹¹¹In-labeled liposomal formulations [16].

Out of various nanoparticles, liposomes have been labeled in different ways, as depicted in Fig. 4a. In the surface radiolabeling, the radionuclide could be attached to the membrane with or without a chelator and PEG chain or could incorporate into the lipid bilayer directly. In intraliposomal radiolabeling, remote and unassisted loading and ionophore-based labeling could be occurred [17].

Fig. 4

The radiolabeling approaches used for vesicular nanomaterials: a liposomes, b niosomes, c extracellular vesicles, and d polymeric nanocapsules

Richardson and coworkers first defined direct radiolabeling without the chelator. They showed that pertechnetate (^99mTcO₄⁻), after reduction to an appropriate oxidation state using SnCl₂ could be directly attached to the surface of liposomes. Presumably, ^99mTc-liposomes were prepared through electrostatic binding to the phosphonate groups in the phospholipid surface of liposomes [18, 19].

Labeling of liposomes without chelator has also been performed using radiofluorinated agents, such as [¹⁸F] FDP (3-[¹⁸F]fluoro-1,2-dipalmitoylglycerol), which could be added during the process of liposome formation [20, 21]. The long alkyl chain in the [¹⁸F]FDP structure might intercalate with the lipid bilayer of the liposomes [22].

Chelator-based radiolabeling of liposomes could be achieved by chelator conjugation on the surface of liposomes. For instance, DTPA-functionalized liposomes (DTPA: diethylenetriamine pentaacetic acid) have been extensively labeled with ^99mTc and ¹¹¹In [23,24,25]. Of note is that the biodistribution of these radiolabeled liposomes may be altered based on the extra radio-complex deposition [26]. Due to this drawback of surface labeling, many researchers attempt to label liposomes inside the liposomal core. This strategy can enhance the stability of the radiolabeled system. Some preliminary studies have been done to encapsulate a DTPA complex into the core of liposomes during liposome preparation [27].

The use of ionophores for transporting the radiolabeled complex across the lipid bilayer of the liposomes is the most practical intra-liposomal radiolabeling approach. The first study for this technique was reported by Gamble et al. They used the calcium ionophore for encapsulating ¹¹¹In into the liposomal bilayer as the nitrilotriacetic acid (NTA) complex [28]. Also, other ionophores (8-hydroxyquinoline; oxine) and chelators (DTPA) were introduced for radiolabeling of liposomes with ¹¹¹In [29].

Based on literature, the presence of ionophore for delivering the radionuclide inside the liposome is not necessary. In a study performed by Henriksen et al., the ⁶⁴Cu²⁺ ions were loaded inside the liposomes entrapping a chelator (DOTA) via unassisted transportation across the lipid bilayer. Using this method, the DOTA-containing liposomes at neutral internal pH were efficiently (RY% > 95) radiolabeled with high stability. No need for potentially toxic ionophores is another positive aspect of unassisted loading method [30].

Van Der Geest et al. compared radiolabeling methods of three formulations of liposomes, including DTPA-liposomes, DTPA-DSPE-liposomes (DSPE: 1,2-distearoyl-Sn-glycero-3-phosphorylethanolamine), and empty liposomes (without DTPA). Radiolabeling yield of these liposomal formulations was investigated at different specific activities. The results showed that radiolabeling efficiency (RE) of DTPA-liposomes and DTPA-DSPE-liposomes was > 95% at a specific activity of 0.15 GBq ¹¹¹In per mmol total lipid. The RE of DTPA-DSPE-liposomes was > 90% at a specific activity of 0.15 to 15 GBq/mmol, but the RE of DTPA-liposomes decreased significantly at specific activity higher than 0.15 GBq/mmol. The RE of empty liposomes was 62% at a specific activity of 0.15 GBq/mmol. Therefore, DTPA-DSPE-liposomes and DTPA-liposomes could be efficiently radiolabeled with ¹¹¹In; however, the specific activity may be a limiting factor for radiolabeling of DTPA-liposomes [31].

Moreover, it has been shown that it is possible to radiolabel liposomes without the encapsulated chelators and chemical modification of the formulation. In this method, the chelating capabilities of some drugs — encapsulated in the liposome with high concentrations — provide a chance to bind the radionuclide after its transporting through the bilayer using an ionophore [32, 33]. The interaction of manganese with hydroxyl and carbonyl groups of the doxorubicin [34] and ⁸⁹Zr with the carboxylate of methylprednisolone hemisuccinate provides suitable sites for radiolabeling [32]. This radiolabeling approach prepared diverse liposomal formulations with different radionuclides as ⁸⁹Zr, ⁶⁴Cu, and ⁵²Mn. However, insufficient strength of the radio-metal bond with the drug may limit the radiolabeling yield using this method [32, 35, 36].

In addition, the liposomes were radiolabeled by remote loading of radiopharmaceuticals inside their cores. A neutral and lipophilic radiopharmaceutical can diffuse into the aqueous core of the liposome in where they can be protonated and then trapped, as an experiment done by Bao and coworkers. They used ^99mTc-complex to radiolabel liposomes already encapsulated with glutathione (GSH). Indeed, GSH was used to reduce the radiopharmaceutical and make it trapped into the liposome, and this method has improved the stability of radiolabeled liposomes in serum compared to the empty radiolabeled liposomes. In this study, BMEDA was used as a chelator for remote loading of liposomes with ⁹⁹Tc and ¹⁸⁶Re [37, 38]. So, this approach requires encapsulation of GSH into liposome structures before radiolabeling.

Unlike, Lee et al. reported the remote radiolabeling of liposomes using ⁶⁴Cu-4-DEAP-ATSC (diacetyl 4,40-bis(3-(N,N-diethylamino)propyl)thiosemicarbazone) complex, without liposomal structure modification. Due to the protonation of tertiary amine groups of the ⁶⁴Cu:4-DEAP-ATSC complex inside the liposomes, the complex can stably be trapped within it. In this way, several radiolabeled liposomal formulations have been prepared with high radiolabeling yield and serum stability [39, 40]. The [¹²⁵I]-ADA and [¹²⁴I]-ADA complexes are other agents for remote radiolabeling liposomes with high serum stability and labeling efficiency. ADA, amino diatrizoic acid, as a membrane-crossing amine, could be radioiodinated, and then, this imaging agent can be remotely loaded into the liposomes via transmembrane pH gradients. Remote loading of ADA was performed using ammonium sulfate and citrate-based pH gradient techniques. Remote loading via the citrate-based pH-gradient method has exhibited higher loading efficiency than the ammonium sulfate-based method [41]. The list of different radiolabeled liposomes is summarized in Table 1.

Table 1 Summary of the radiolabeled liposomes

Niosomes

Niosomes are multilamellar and non-ionic surfactant-based vesicles that can incorporate hydrophobic or hydrophilic drugs in their construction. Niosomes have attractive advantages such as high stability, good biocompatibility, biodegradability, and controlled drug release, making these nanosystems a promising platform for drug delivery [59]. Furthermore, niosomes have advantages over liposomes, such as higher chemical stability of surfactant than phospholipid, higher stability in storage, fewer purity problems, and lower manufacturing cost [60].

Radiolabeled niosomes can be applied to imaging diseases such as cancer, infection, and inflammation. Moreover, the pharmacokinetic and biodistribution of niosomes can be investigated using radiolabeling of niosomes [61]. Radiolabeling of nanoparticles such as niosomes allows for tracking nanocarriers using nuclear medicine imaging techniques such as PET and SPECT. ^99mTc as a radionuclide can determine the accumulation and biodistribution of nanoparticles in vivo. ^99mTc has been extensively used for labeling nanoparticles in the field of diagnostics because of their unique physical and chemical characteristics [62]. In a study conducted by Silva et al., niosomes were labeled with ^99mTc through surface chelation using DTPA chelator, and then, radiolabeling yield of ^99mTc-labeled niosomes was optimized. For example, the optimized pH and incubation time for obtaining high RY were obtained 5 and 15 min, respectively [63]. Moreover, Almasi and coworkers investigated the biodistribution and pharmacokinetic of the various components of niosomes through radiolabeling with ^99mTc-HMPAO complex [61]. There are a few examples of radiolabeled niosomes described in Table 2. The radiolabeling methods used for niosomes have been illustrated in Fig. 4b as well.

Table 2 Summary of the radiolabeled niosomes

Exosomes/extracellular vesicles

Extracellular vesicles (EVs) are defined as nano-sized phospholipid-based spherical proteolipids released from various cells, such as macrophages, dendritic, cancerous, and stem cells. Also, EVs may be present in different physiological fluids [64, 65]. Exosomes provide a suitable platform for clinical applications due to their unique characteristics like non-toxicity, non-immunogenicity, biodegradability, and targeting ability [66]. Recently, EVs, in particular exosomes, have attracted much attention from various research fields, such as anticancer or mRNA drug delivery approaches and regenerative medicine in diseases with tissue loss. It is presumably due to their biological origin. Thus, more study on the biodistribution of EVs seems to be essential [64, 67,68,69].

Many studies have been performed to investigate the in vivo biodistribution of EVs using optical imaging. However, optical imaging faces limitations in light penetration into deep tissues [70, 71]. Non-invasive nuclear imaging using radiolabeled EVs can be a good alternative for evaluating the pharmacokinetics of EVs, though. So, clinical studies on EVs as drug delivery systems could be facilitated by monitoring their biodistribution and understanding the fate of EVs [71, 72].

There are several approaches for radiolabeling of EVs. Although, most of them have reported chelator-based radiolabeling in which chelator can be conjugated to the phospholipids or protein on the surface of EVs.

This radiolabeling method could offer a rapid and straightforward radiolabeling technique. The type of chelator and radionuclide is chosen based on the radionuclides’ applications as a diagnostic agent (SPECT, PET tracer) or therapeutic or theranostic agent [4, 73]. In this context, Shi et al. conjugated the NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) as a chelating agent to the EVs surface through free amine groups on their membrane [74].

The existence of protein on the surface of EVs provides a chance for direct radioiodination via electrostatic substitution of iodine on the tyrosine side chain. In these cases, the high uptake of radioactivity in the thyroid gland, indicating the release of iodide, has been reported. So, labeling EVs with radioiodine is not preferred compared to other radiolabeling techniques [75, 76]. In this context, a study reported quantification of the in vivo biodistribution of ¹³¹I-labeled EVs from diverse cellular origins. Figure 5 (left) indicates in vivo SPECT/CT imaging in the animals, which applied for detection and quantification of ¹³¹I-labeled tumor cell-derived EVs in the primary metastatic site and tumor. Figure 5 (right) shows 3 h after intravenously injection of the ¹³¹I-labeled exosomes to tumor-bearing mice, EVs from all the groups accumulated in the primary tumor and metastatic sites except for the HEK293 exo [76].

Fig. 5

SPECT/CT imaging after injection of 350 µCi of ¹³¹I-labaled EVs (It has been adapted and reproduced with permission from ref. [76].)

Chelator-free direct radiolabeling of EVs with high radiolabeling yield and serum stability has been performed in some studies. As a simple method, radionuclides can be trapped into the EVs structures. For instance, Jang et al. labeled exosome-nanovesicles collected from the murine macrophage cell lines using ^99mTc-HMPAO. HMPAO, with lipophilic nature, can pass through the lipid bilayer of the cellular membrane. Then, it is reduced and got more hydrophilic in the presence of GSH and cannot escape from the lipid bilayer. The presence of GSH into the EVs makes the labeled EVs stable until the integrity of lipid bilayers is maintained [71, 77].

EVs could label using radio-ionophore due to the presence of lipid bilayer in their structure. But, the lower serum stability and radiolabeling yield are its main drawback compared to surface radiolabeling using a chelator [64].

Furthermore, EVs have been radiolabeled through remote loading with high efficiency. However, this method demonstrated uptake in the salivary gland indicating the release of ^99mTc [78].

Figure 4c demonstrates the radiolabeling methods of extracellular vesicles (EVs), including membrane radiolabeling, intraluminal labeling, and covalent binding. Table 3 lists several examples of radiolabeled EVs.

Table 3 Summary of the radiolabeled exosomes

Polymeric micelles and polymeric nanoparticles

Polymeric micelles (PMs) are constructed via the self-assembling of amphiphilic block copolymers. PMs with hydrophilic shells and hydrophobic cores extensively served as nanocarriers in biomedical applications to encapsulate hydrophilic and hydrophobic cargo [83, 84].

There are several methods for radiolabeling PMs to determine their in vivo fate (Fig. 4d). Direct radiolabeling of polymeric micelles with ^99mTc has been reported in several studies [85, 86]. Furthermore, radioiodination of PMs is possible, but this technique requires labeling the polymer before micelle preparation and a longer time limiting its applications [87, 88]. Moreover, the researchers also radiolabeled polymeric micelles using chelator-based methods [89]. Different examples of radiolabeling PMs are summarized in Table 4.

Table 4 Summary of radiolabeled polymeric micelles and polymeric nanoparticles and nano-capsules

Polymeric nanoparticles (PNPs) — including nanocapsules and nanospheres — are another nano-sized solid polymeric particles [100]. The nanocapsules have vesicular structure, while the nanospheres render a matricial organization of the polymeric chains. PNPs have been radiolabeled using various techniques which is suitable for each structure. Such PNPs as poly lactic-co-glycolic acid (PLGA) NPs and poly lactic acid (PLA) NPs have been directly radiolabeled with ^99mTc [101, 102]. Chitosan-based nanomaterials could also be radiolabeled with ^99mTc through binding to the hydroxyl and amine groups of chitosan [90, 103]. In addition, (radio)halogenation-based reactions have been used in several studies for radiolabeling of PNPs. This method requires structure modification of PNPs, though. For example, N-(2-hydroxypropyl)-methacrylamide (HPMA) polymeric nanoparticles modified with tyramine were radiolabeled using 2-[¹⁸F]fluoroethyl-1-tosylate ([¹⁸F]FETos) prosthetic group [104, 105].

However, the specific structure of PNPs can be radiolabeled through a radioiodination reaction without structural modification. For instance, the presence of phenol residues on polyvinyl phenol (PVP)–based NPs or phenol-containing polymethyl methacrylate (PMMA) in the PLGA-PMMA copolymer NPs provides radiolabeling sites [106, 107]. Polymeric structures can be conjugated to the chelators for indirect radiolabeling. A related study was radiolabeling of cellulose-based nanoparticles with ¹¹¹In using bioconjugates. In this study, the terminal aldehyde group or the hydroxyl groups on the backbone of cellulose has interacted with DO3A-hydrazine and DO3A amine chelators (DO3A: 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl), respectively The presence of more chelators for the hydroxyl-conjugated NPs after reaction with ¹¹¹In provided higher radiochemical yield than aldehyde-conjugated NPs [108]. Moreover, [^99mTc]-HMPAO complex could be trapped into PLA nanocapsules during the synthesis of NPs [109]. Figure 5d represents the radiolabeling methods of polymeric nanocapsules including loading of radionuclides into their vesicular core, and surface radiolabeling. In addition, the methods used for radiolabeling of polymeric micelles as nucleated organic nanoparticles are illustrated in Fig. 5c.

Protein-based nanoparticles

Protein-based nanomaterials are constructed from proteins as precursors self-assembling into the 3D-nanoparticles [110]. Protein-based nanoparticles have exhibited excellent biocompatibility [110], biodegradability, amphiphilicity required for interaction with drugs [111], and target ability [112]. The Intrinsic biological origin of proteins makes them promising nanostructures for biomedical applications [113].

An example of a clinical-approved protein-based nanomaterial is albumin-bound paclitaxel (Abraxan), composed of human serum albumin (HSA) nanostructure and paclitaxel as a chemotherapeutic drug that could be used for tumor therapy [114]. As the most abundant plasma protein, albumin is a suitable protein-based carrier for steroids, lipophilic hormones, and lipophilic drugs [115]. Similar to the other nanomaterials discussed previously, the determination of biodistribution and pharmacokinetics of protein-based nanoparticles is helpful for further biomedical and clinical studies. In this regard, various studies have been reported for radiolabeling and imaging of protein-based nanoparticles in vivo (Fig. 6a) [4].

Fig. 6

PET/CT imaging after injection of 0.23 mCi of [⁶⁴Cu]-labeled dendrimer in male mice bearing PSMA⁺ PC3 PIP and PSMA⁻ PC3 flu tumors (It has been adapted and reproduced with permission from ref. [116].)

Radiolabeling of protein-based nanoparticles has been mostly performed for serum albumin NPs. SA NPs could be directly radiolabeled with ^99mTc using SnCl₂ with high radiolabeling yield and stability in a buffer [117]. Moreover, radiolabeling of SA nanoparticles has been carried out through direct radioiodination of tyrosine residues on SA nanoparticles using ¹²⁵I for SPECT/CT imaging and ¹³¹I for therapeutic purposes [118]. Radiolabeling of SA NPs using chelators is another strategy that could be used. For example, DTPA served as a chelator for radiolabeling SA nanoparticles with ¹¹¹In. DTPA can covalently bind to the surface of SA particles by introducing amine groups into the matrix of NPs [119, 120]. Polypeptide-based nanoparticles are also a kind of protein-based nanomaterials that can be labeled with ^99mTc via their His-tags. His-tag usually includes six to nine histidine residues at the N- or C-terminal of peptides which can serve as a tridentate ligand for complex formation with tricarbonyl technetium core. The coordinate bonds between His-tag and tricarbonyl technetium showed high in vivo stability (Fig. 6a) [121]. High-density lipoprotein-based nanoparticles have been radiolabeled with ⁸⁹Zr using DFO as a chelator [122, 123]. Table 5 lists the radiolabeled protein-based nanoparticles.

Table 5 Summary of the radiolabeled protein-based nanoparticles

Dendrimer

Dendrimers are nanostructured macromolecules containing three domains, a core at the center of the structure, branches composed of repeating units, and many terminal groups [129]. The biocompatibility and tunable structure of dendrimers make them promising platforms for biomedical applications such as drug delivery [129]. Moreover, the structure of dendrimers and their surface modifications provide radiolabeling for the investigation of their biodistribution and pharmacokinetics. Most radiolabeling studies have been performed on poly(amidoamine) (PAMAM)-based dendrimers. The most common radiolabeling technique of PAMAM is conjugation of a chelator to amine residues on the PAMAM polymer. For instance, PAMAM dendrimers have been radiolabeled with ¹¹¹In using DTPA [130,131,132,133]. In addition, they have been labeled with ¹⁷⁷Lu using DOTA-based conjugated such as BFCs DOTA-NHS (DOTA: (1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid) [134]. The DOTA-NHS, as a bifunctional chelating agent, was also conjugated to the PAMAM and labeled with ⁶⁴Cu [116, 135]. In a study, Lesniak et al. compared the radiolabeling quality and biodistribution of dendrimers labeled with ¹¹¹In and ⁶⁴Cu using DOTA. The percentage of radiolabeling yield for both nanoparticles was approximately 80%. During 48 h post-injection of radiotracers, the liver uptake of ⁶⁴Cu-labeled dendrimer was 6–eightfold higher than the ¹¹¹In-labeled ones. Unlike ¹¹¹In-labeled NPs, the spleen uptake for ⁶⁴Cu-labeled NPs was decreased from 1 to 48 h post-injection. These results indicated that ⁶⁴Cu was presumably released from dendrimers. PET/CT images after injection of these ⁶⁴Cu-labeled dendrimers are demonstrated in Fig. 6 [116].

Moreover, it is likely due to the better binding energy of DOTA for chelating ¹¹¹In than ⁶⁴Cu. Interestingly, it has been shown that NOTA is a more favorable chelator for ⁶⁴Cu [136].

Additionally, the amine moieties on the PAMAM served as radioiodination sites using amine-reactive reagents such as Bolton–Hunter reagent [137, 138] and N-succinimidyl 3-iodobenzoate [139, 140].

PAMAM dendrimers have also been radiolabeled with radionuclides directly. In this regard, the radionuclide is bound to the amine groups on the PAMAM dendrimers. For example, direct labeling of PAMAM in treatment by [^99mTcO₄]⁻ has been performed in several studies using SnCl₂ reductant with high efficiency and stability [141, 142]. Some examples of the radiolabeled dendrimers are summarized in Table 6. These radiolabeling approaches are also indicated in Fig. 7b.

Table 6 Summary of the radiolabeled dendrimers

Fig. 7

The radiolabeling approaches used for nucleated organic nanoparticles: a protein-based nanoparticles, b dendrimers, and c polymeric micelles

Inorganic nanomaterials

Inorganic nanoparticles are a large family of nanomaterials ranging from metal, metal oxide, and lanthanide-doped upconversion nanoparticles (UCNPs) to semiconductor nanocrystals (quantum dots) with attractive physicochemical properties [149]. X-ray absorption, energy transfer, heat, and reactive oxygen generation are some of their characteristics [150]. These materials are prone to be radiolabeled and provide appropriate scaffolds with dual functions as theranostic agents or dual-modality imaging agents due to the presence of metal in their structures [149].

Silica nanoparticles

Silica nanoparticles (SNPs) are biocompatible particles with high stability and some practical applications. Solid silica nanoparticles (SSNPs) and mesoporous silica nanoparticles (MSNs) are two main groups of these particles [151]. Bio-responsive properties and the ability to adjust their mesoporous pore size make them appropriate carriers for controlled drug delivery [152].

Unlike other inorganic nanomaterials, SNPs do not have optical or magnetic properties. However, they have often been used in different studies because of their easily adjustable textural features, including porosity, crystallinity, and morphology [153]. Thanks to chemical groups on their surfaces, SNPs can be functionalized with various molecules affecting their drug loading, active targeting, and behavior in the body [154]. High pore volume for drug encapsulations, low cost, and simple synthesis are the other advantages of these particles [155].

Due to the safety of oral administration of silica has been approved by the FDA since 2010, the chance of silica nanoparticles for clinical studies is very high [156]. In this regard, evaluating their pharmacokinetics and biodistributions using radiolabeled SNPs can be helpful.

The presence of silanol groups (–Si–OH) on the surface of SNPs is a valuable site for radiolabeling of silica nanoparticles owing to the high affinity of oxophilic cations, such as ¹¹¹In, ⁸⁹Zr, to these groups. Radiolabeling of SNPs using this technique is easy, fast, and robust [157].

Comparing the stability of radiolabeling methods indicated that radiolabeled SNPs using chelators are more stable than chelator-free ones. Radiolabeling of cRGDY-conjugated fluorescent silica nanoparticles (C dots) with ⁸⁹Zr in the presence of DFO, as a chelating agent demonstrated more stability than radiolabeling without DFO. More than 25% of chelator-free radiolabeled silica nanoparticles are released free ⁸⁹Zr after 48 h, while this percentage for chelator-based radiolabeled nanoparticles was lower than 2% at the same time [158].

There are different chelator-free strategies for radiolabeling of SNPs, including chemical adsorption of radiometals, using radioiodination mediators (Bolton–Hunter reagent), and ^99mTc reduction using SnCl₂ and His-tag. Out of them, the chemical adsorption of radiometals like ⁶⁴Cu, ⁹⁰Y, and ⁶⁸Ga on the SNPs is the best choice due to its rate of radiolabeling reaction, stability, and simplicity [4].

The radiochemical stability of labeled SNPs depends on the oxophilic nature of the radionuclide. In vivo PET imaging revealed that the radiolabeled SNPs with a dense radionuclide such as ⁶⁸Ga and ⁸⁹Zr possessed high stability. Radiolabeling SNPs using ⁶⁴CU as a softer radionuclide exhibited inferior stability with 50% radionuclide leakage after 4 h, though [157]. The functionalization of silica nanoparticles with thiol groups could improve SNPs radiochemical stability with ⁶⁴Cu and increase their stability from 35 to 90%. Of note is that labeling of MSNs with ⁶⁴Cu is temperature-dependent, so labeling at a temperature > 70 °C seems to be essential for their stability. For example, radiolabeling of silica nanoparticles at 37 °C with radiolabeling yield greater than 85% has shown the release of free radionuclides from nanoparticles in serum [159]. Coating radiolabeled nanoparticles can also be another practical approach for improving their stability. ⁸⁹Zr-labeled hollow mesoporous silica nanoparticles (HMSNPs) without chelator, which were coated with the membrane of red blood cells, exhibited higher stability than uncoated nanoparticles due to avoiding the release of ⁸⁹Zr. Furthermore, this coating prevents the phagocytosis of nanoparticles and prolongs their biodistribution [160].

Radiolabeling of SNPs with special radionuclides under different conditions showed that radiolabeling yield was higher than 99% at 70 °C. So, a high temperature was required for the complex formation of SNPs. However, the pH alteration did not significantly affect it [157]. In addition, the radiolabeling yield depends on the incubation time, so a longer reaction time increases the probability of radiolabeling. For instance, the radiolabeling yield of ⁸⁹Zr-labeled HMSNPs interacted with human red blood cell membranes was 81% at first 30 min, and it was increased to 96% after 24 h incubation time [160].

Radiolabeling of MSNPs with ⁶⁴Cu served as PET imaging and biodistribution assessment on breast cancer-bearing mice model. In this study, the PEGylated SH groups and NOTA were used for the functionalization and radionuclide chelating of SNPs (Fig. 8a). The best radiolabeling conditions were achieved at 37 °C and pH 6.5 during the 30-min incubation. PET imaging also revealed that the complex was stable (> 90%) for up to 48 h [161]. Some of the other radiolabeled SNPs have been summarized in Table 7.

Table 7 Summary of the radiolabeled silica nanoparticles

Fig. 8

Radiolabeling approaches used for a silica nanoparticles, b gold nanoparticles, c CuS nanoparticles, and d upconverting nanoparticles. Part a has been adapted and reproduced with permission from ref. [162]

Gold nanoparticles

Gold nanoparticles are promising nanocarriers for biomedical applications. Due to the presence of the thiol groups, these particles are stable and biocompatible, being of adjustable size, large surface, and high affinity to the organic molecules [162].

The optical properties of gold nanoparticles make them appropriate to assay the intracellular living events without any incision in the body, which is useful for diagnosing disease and targeting drug delivery systems [162]. These particles also have the ability for surface-enhanced Raman scattering (SERS) and X-ray absorption by modifying their particle size, charge, and shape. Therefore, they are desirable platforms for multimodal imaging such as CT, MRI, and radionuclide imaging [150].

The slow elimination and tissue accumulation of gold nanoparticles may cause inflammation, apoptosis, and toxicity. Thus, precise pharmacokinetics evaluation seems to be essential, and radiolabeling of these nanoparticles contributes to the purpose of this assessment [150]. Moreover, the photothermal properties of gold nanoshells enable them to destroy cancer cells [171], and monitoring their biodistribution is supportive for precise photothermal ablation of small cancerous cells [162].

Some modifications to the shape and morphology of gold nanoparticles have been performed to make them ready to radiolabel with a wide range of radionuclides [4]. Gold nanoparticles have been mostly radiolabeled with ⁶⁴Cu (chelator-based or chelator-free strategies) and ^99mTc using chelating agents [4].

The radiolabeling yield of thiol-capped gold nanoparticles with ⁶⁴Cu was more than 94% using DOTA-maleimide at pH 8.8 and different temperatures ranging from room temperature to 60 °C. The released ⁶⁴Cu from gold nanoparticles was lower than 4% in the presence of EDTA. PET imaging was applied to assess the nanoparticles’ biodistribution in healthy mice. Figure 9 demonstrates the PET imaging after IV and oral administration of radiolabeled gold nanoparticles at different time points [172].

Fig. 9

MicroPET imaging after IV (100 µCi) and oral administration of radiolabeled thiol-capped gold nanoparticles at different time points (It has been adapted and reproduced with permission from ref. [172].)

A dynamic PET imaging after injection of glutathione-coated ultra-small gold nanoparticles radiolabeled with ¹⁹⁸Au or ⁶⁴Cu showed that ⁶⁴Cu–NOTA–Au–GS was rapidly excreted via the renal system for 6 min [173]. PEGylation of these nanoparticles or decreasing their density could reduce the clearance rate of these nanoparticles though [174, 175].

The bombesin-targeted ^99mTc/¹⁷⁷Lu–AuNP was also successfully used for SPECT imaging and radiation therapy of prostate cancer. In this regard, ^99mTc with the aid of hydrazinonicotinyl-Tyr3-octreotide (HYNIC-TOC) and ¹⁷⁷Lu using DOTA-Gly-Gly-Cys were attached to the gold nanoparticles [176].

Technetium-99 m could label gold nanoparticles with high efficiency. Briefly, the gold nanoparticles were functionalized with HYNIC-GGC and thiol-mannose group and then labeled with ^99mTc and used for sentinel lymph node scintigraphy (Fig. 8b). These radiolabeled nanoparticles exhibited high stability so that only 1% of free ^99mTc (pertechnetate) was released after 24 h incubation with fresh human serum at 37 °C [177].

The zirconium-89 was also applied to radiolabeling gold nanoparticles as a good PET imaging agent in the mice models. In this case, cetuximab as a targeting agent against epidermal growth factor receptor (EGFR) was functionalized with chelating agent desferal and radiolabeled by ⁸⁹Zr (> 75% at ambient temperature after 60 min), and then, the complex was conjugated to the gold nanoparticles [178].

The hot-plus-cold is a convenient method of chelator-free radiolabeling strategies applied for gold nanoparticles. In this strategy, a mixture of reagents containing the radionuclide (hot precursor) and the non-radioactive nanomaterial precursors (cold precursors) is reacted to make the radiolabeled nanoparticles. When the radionuclide is added to the nanoparticle precursors during the synthesis, co-precipitation process is performed. This leads to the incorporation of the radionuclide into the structure of the nanomaterial. The selected radioisotopes (¹⁹⁸Au, ¹⁹⁹Au, ¹¹¹In, and ⁶⁴Cu) could be well embedded into the crystal structure of final nanocrystals, resulting in intrinsically radioactive nanoparticles with higher stability compared to chelator-based methods [4]. For instance, radiolabeling of gold nanoparticles with ⁶⁴Cu using the hot-plus-cold method showed high stability in mouse serum over 48 h without degradation. Indeed, ⁶⁴CuCl₂ and gold chloride copper acetylacetonate could interact together as hot and cold precursors, respectively [150].

The main drawbacks of radiolabeled gold nanoparticles are their poor in vivo stability and non-efficient active targeting. Such methods as PEGylation, coating, and surface functionalization have been introduced to improve their stability and targeting ability [150]. Sun et al. demonstrated that the PEGylated gold nanoparticles were radiolabeled with ⁶⁴Cu using hydrazine as a radionuclide reducing agent in the presence of polyacrylic acid. The radiolabeling yield of this reaction was 100% with using hydrazine as a reduction agent, and reduction was 30% without using it. Stability was high, and leakage of ⁶⁴Cu was lower than 3% in PBS after 24 h. Arginine-glycine-aspartic acid (RGD) peptide for targeting U87MG glioblastoma xenograft model was used for theranostic applications of these labeled nanoparticles for PET imaging and photothermal cancer therapy [179]. The other examples of gold nanoparticles radiolabeled with different radionuclides have been summarized in Table 8.

Table 8 Summary of the radiolabeled gold nanoparticles

Copper sulfide nanoparticles

As stable and biocompatible particles, copper sulfide nanoparticles (CuS NPs) can absorb NIR and convert it to thermal energy that is useful for the destruction of cancer cells. These nanoparticles can be served as imaging and photothermal therapeutic agents concurrently [150]. The literature has often applied the chelator-free hot-plus-cold method (type I) for ⁶⁴Cu radiolabeling of CuS NPs (Fig. 8c). Simple interaction between ⁶⁴CuCl₂ and a mixture of CuCl₂ and Na₂S produces the radioactive ⁶⁴CuS nanoparticles [189]. Due to the formation of ⁶⁴Cu atoms inside the typical nanocrystals from the beginning of the synthesis process, personal exposure is presumably higher in condition that fast and appropriate purification is not performed afterwards. This scaffold containing ⁶⁴Cu also provides a prudent agent with low activity and high stability for PET imaging [150]. Zhou et al. synthesized the ⁶⁴CuS nanoparticles with a high percentage yield using the “hot-plus-cold” method and coated them with citrate and polyethylene glycol (PEG) to improve their stability. Radiochemical stability test revealed that PEGylated ⁶⁴CuS nanoparticles were more stable than citrate-coated ones [190]. Zhou et al. used the bovine serum albumin (BSA) template as a medium for chelator-free radiolabeling of CuS NPs with ⁶⁸ Ga. In this system, the Mn²⁺ ion was also doped on the surface of CuS NP to provide MRI properties. The prepared nanoparticles were studied for photothermal therapy and triple-modal imaging, including MRI, PET, and photoacoustic tomography (PAT) [191]. Chelator-based labeling is another approach for CuS NPs radiolabeling which is shown as method type II in Fig. 8c. Chen et al. synthesized water-soluble CuS NPs encapsulated in mesoporous silica shells using positively charged cetyltrimethylammonium chloride (CTAC). To radiolabel these nanoparticles with ⁶⁴Cu, the NOTA chelating agent was attached to the amino groups on their surfaces. The final⁶⁴Cu-CuS-MSN was introduced as a desirable theranostic nanoparticle [192]. Table 9 summarizes the researches performed on radiolabeling of CuS nanoparticles.

Table 9 Summary of the radiolabeled CuS NPs

Upconverting nanoparticles

Upconverting nanoparticles (UCNPs) can convert two or more low-energy photons to a single high-energy photon with unique optical features. These particles can be excited by NIR rays, and consequently, they emit light in the ultraviolet and visible regions. Due to their ability, they have useful applications in deep tissue imaging, fluorescent microscopy, and nanomedicine [4].

Some reports in the literature have shown their potentiality in medical imaging. For instance, the ⁶⁴Cu-labeled UCNPs coated with porphyrin-phospholipids have been developed as a hexa-modal imaging agent. The nanoparticles were quickly and efficiently (radiochemical purity > 80%) labeled with ⁶⁴Cu using porphyrin and utilized for imaging via fluorescence, upconversion, positron emission tomography, computed tomography, Cerenkov luminescence, and photoacoustic tomography [199].

Seo et al. also demonstrated admirable radiochemical stability for ⁶⁴Cu-UCNPs labeled using NOTA as a chelator and coated with micelles. PET imaging exhibited that the radionuclides did not detach from the nanoparticles after long-term passage through the hepato-biliary tract system in the mice models. Of note is that this finding was reinforced with luminescence imaging ex vivo [200].

UCNPs can be directly labeled with ¹⁸F using anion (¹⁸F⁻) anchoring and isotopic exchange methods. In other words, no prosthetic group was used for radiolabeling of them. The former strategy is the result of a reaction between UCNPs as a subgroup of rare-earth cations and ¹⁸F⁻. The final product was easily purified using aqueous washing and centrifugation. The reaction yield was greater than 90% at room temperature within a short time. A noteworthy feature of this method was that ¹⁸F-labeled SiO₂-coated UCNPs did not show obvious radioactivity, indicating the high surface area and pore volume of SiO₂ had no significant effect on radiolabeling and only specific interaction between ¹⁸F⁻ anions and UCNPs as rare-earth cations are important. These particles were used for biodistribution assessment and PET imaging in mice models. The radiochemical stability test on ¹⁸F-labeled UCNPs revealed that ¹⁸F dissociation from rare-earth nanoparticles such as hydroxide and fluoride was lower than from the oxide [201]. Homogeneous isotopic exchange is a simple, effective, and robust strategy for radiolabeling UCNPs with ¹⁸F directly. Isotopic exchange can occur between ¹⁹ and ¹⁸F at room temperature quickly. For instance, ¹⁸F was used for radiolabeling of NaYF₄ with high efficacy (> 92%) [4]. Table 10 lists the radiolabeled UCNPs which have been reported yet. Figure 8d illustrates the approaches used for radiolabeling of upconverting nanoparticles.

Table 10 Summary of the radiolabeled CuS NPs

Quantum dots

Quantum dots (QDs) are crystal nanoparticles (2–10 nm diameter) composed of groups II to VI or III to V elements. Out of QDs, cadmium selenide (CdSe) capped with water-soluble agents is widely used in experiments [150]. These particles are semiconductors with innate fluorescent capability for drug delivery and bioimaging [4]. Unlike organic dyes or fluorescent proteins, QDs are photostable and can emit different photons from visible to infrared wavelength ranges depending on their size and composition [150]. Passive targeting and tumor accumulation via the EPR effect are possible using QDs as well [205]. Considering the limitation of optical imaging as poor tissue penetration, using the radiolabeled QDs for SPECT and PET imaging can be helpful and has been widely studied in the literature. Some of them are pointed out here and depicted in Fig. 10a [150]. Hot-plus-cold precursor’s strategy and heterogeneous isotopic exchange are helpful chelator-free methods for radiolabeling of QDs. There are many experiments on ⁶⁴Cu and ⁶⁸ Ga labeling of QDs using chelator-free methods. As Tang et al. reported, the labeling yield and radiochemical stability (for up to 24 h) of ⁶⁸Ga and ⁶⁴Cu-labeled ZnS dots using the heterogeneous isotopic exchange method was higher than 90% [4]. Guo et al. also studied chelator-free radiolabeling CIS/ZnS QDs with ⁶⁴Cu, and they found that the complex was stable enough [206]. On the other hand, the radiochemical stability of chelator-based radiolabeling QDs is still challenging [150]. For example, Felber et al. prepared a ^99mTc-labeled CdSe/ZnS core–shell QDs with high radiochemical purity (> 95%). To radiolabel these QDs, the 2,3-diaminopropionic acid (DAP) was utilized as a strong chelator for [^99mTc(CO)₃]⁺ fragment. Of note is that DAP was anchored to the surface of QDs using thiol groups and PEG linker. Nonetheless, this tracer was unstable in serum, and HS-PEG-DAP was partially detached from the nanoparticle after 1 week [207].

Fig. 10

The radiolabeling approaches adopted for: a quantum dots, b magnetic nanoparticles, c graphene oxide nanoparticles, and d metal–organic frameworks. Part c has been adapted and reproduced with permission from ref. [4]

Such parameters as the amount of QDs, incubation time, and temperature are critical in complex formation and radiolabeling yield. For instance, the radiolabeling of the amino- or carboxyl-functionalized QDs was performed using [¹⁸F]fluoroethyl tosylate and [¹¹C]methyliodide as prosthetic groups. These particles were used for fluorescence and PET imaging. The results revealed that the yield of a reaction in amino-QDs was higher than carboxyl ones. For both of them, high radiolabeling yields were obtained at 120 °C. The yield of ¹¹C labeling in both functionalized-QDs was greater than ¹⁸F labeling (~ 40% vs. 5%) because [¹⁸F]fluoroethyl tosylate is not a suitable agent for reaction in the aqueous system [208].

In spite of that radiolabeling of QDs with fluorescence emission properties is helpful for their biological applications, special attention to the toxicity of these particles is an important issue. Cadmium as a core of QDs has been widely used in many types of research for radiolabeling yet. Although, their toxicity problem and heterogeneous biodistribution make them unappropriated for in vivo application. Biocompatible metals may be a more suitable alternative for clinical usage of the radiolabeled QDs, though [1]. Some of the radiolabeled QDs nanoparticles are summarized in Table 11.

Table 11 Summary of the radiolabeled QD nanoparticles

Magnetic nanoparticles

Magnetic NPs (MNPs) are particles able to respond to a magnetic field and enhance proton relaxation time in body tissues that can be used as contrast agents for magnetic resonance imaging (MRI) and photothermal therapy. Superparamagnetic iron oxide NPs (SPIONs), paramagnetic gadolinium (Gd), and manganese (Mn) are the three main categories of MNPs. Radiolabeling of MNPs can provide dual-modality imaging agents with therapeutic capabilities for the precise assessment of physiological tissue changes and their treatment concurrently [150].

Like the other nanoparticles, MNPs can also be radiolabeled using chelator-based and chelator-free methods shown in Fig. 10b. Chelator-based is the critical strategy for radiolabeling iron oxide nanoparticles (IONPs) [4]. Technetium-99 m, as a well-known radionuclide, has been mostly used for labeling of IONPs, which can interact with COO⁻ and NH₂ groups of chelating agents [217]. For instance, ultra-small superparamagnetic iron oxide nanoparticles (USPIONs) functionalized with PEG-bisphosphonate were radiolabeled using bisphosphonate dipicolylamine-alendronate (DPA-ale) chelator and applied for SPECT imaging [218].

Chemisorption)chemical absorption( is a chelator-free method for radiolabeling nanoparticles with donor sites on their surfaces. Magnetite (Fe₃O₄) surface can directly form coordination bonds with metallic radionuclides [4]. Chen et al. described the ⁷²As-labeling of IONPs by chemisorption method for PET imaging and biodistribution study in mice models [219]. Feraheme/ferumoxytol nanoparticles were also radiolabeled with ⁸⁹Zr, ⁶⁴Cu, and ¹¹¹In by chemical absorption method with radiolabeling yields ranging from 66 to 93% and radiochemical stability > 90% in human plasma [4]. Due to the high affinity of Al(OH)₃ for F, Cui et al. used Al(OH)₃ as a coating layer on the surface of IONPs for radiolabeling with ¹⁸F. Despite good efficacy for this reaction (> 97%), PET/MRI imaging demonstrated bone accumulation of ¹⁸F⁻ indicating their poor radiochemical stability [150]. Heterogenous isotopic exchange is another approach for efficiently radiolabeling of IONPs. Long incubation time and harsh temperature are required for this reaction, which may impact the physicochemical properties of magnetic nanoparticles, though. So, the other strategies seem to be more suitable for radiolabeling of them, like the “hot-plus-cold” method [220]. For example, the radiolabeling yield for dextran-coated IONPs with ⁶⁸Ga was > 90%. FeCl₃(6H₂O) as a clod and [⁶⁸Ga]GaCl₃ as a hot precursor have been used for the reaction [221]. Radiolabeling of IONPs — a subgroup of MNPs — has been performed with various radionuclides using different methods, as summarized in Table 12.

Table 12 Summary of the radiolabeled IONPs

Graphene/carbon-based nanoparticles

Graphene is the lattice-stacked layer of carbon atoms that produce a single layer of graphite. These nanomaterials with attractive optical and mechanical properties have been used for drug delivery and biomedical imaging. Radiolabeling of graphenes like graphene oxide (GO) and carbon nanotubes (CNTs) provides an opportunity for fluorescent/nuclear imaging concurrently [4]. It is noteworthy that the use of radio-graphene reduces the dose of nanoparticles owing to the high sensitivity of nuclear imaging systems [150].

In chelator-based radiolabeling, amine-functionalized CNTs have been utilized to conjugate the DOTA chelating agent suitable for ⁸⁶Y and ¹¹¹In labeling. These radiolabeled scaffolds were used to assess tissue distribution and pharmacokinetic studies in mice models [227].

Moreover, several functional groups, such as carboxylate at the surface of GOs, make them appropriate for chelator-based radiolabeling [150]. For instance, in the report of Cornelissen et al., the GOs were efficiently labeled with ¹¹¹In. In this case, the p-SCN-Bn-DTPA as a bifunctional chelator was firmly attached to the GOs structures via π-bond interactions [228].

In another study, ⁶⁴Cu-NOTA complex was loaded in the PEG-modified reduced graphene oxide (RGO) with high labeling efficiency, but the mice serum stability test of the mentioned complex was lower than NOTA-conjugated RGO-PEG [4].

Shi et al. applied a new strategy for directly labeling GO and RGO. In this method, the free electrons on the surface of graphene interact with radiometal ions like ⁶⁴Cu²⁺ and ⁶⁸Ga³⁺. Although, the radiolabeling yield and stability of ⁶⁴Cu-labeled RGO were higher than GO due to the higher abundance of π electrons on the surface of RGO compared to GO scaffolds [229, 230]. The other examples of radiolabeled graphene/carbon-based nanoparticles are reported in Table 13. Figure 10c also illustrates the radiolabeling strategies as mentioned above.

Table 13 Summary of the radiolabeled graphene/carbon-based nanoparticles

Metal-organic frameworks

Metal–organic frameworks (MOFs) are crystalline porous materials built from metal ions and organic linkers [237]. Nanoscale metal–organic frameworks (NMOFs) have been extensively used in biomedical applications, such as drug delivery. It is likely due to their attractive characteristics, including high porosity and surface area, biodegradability, biocompatibility, and ease of surface modifications [238, 239]. NMOFs could be used in such various imaging techniques as computed tomography (CT) [240] and magnetic resonance imaging (MRI) [241]. Compared to these imaging modalities, nuclear imaging probes (PET and SPECT) provide more accurate images of deeper lesions. [242]. Examples of the radiolabeled NMOFs used for PET or SPECT imaging are presented in Table 14. The schematic illustration of radiolabeled metal–organic frameworks is demonstrated in Fig. 10d as well.

Table 14 Summary of the radiolabeled MOFs

Commercial and FDA-approved radiolabeled nanoparticles

Commercialization and approval of nanoparticle-based radiopharmaceuticals require collaboration between industry and academic institutes [246]. The accurate design of nanoparticles and their precise toxicological assessment can facilitate the clinical studies of novel nanoparticles as imaging agents [247]. According to the satisfactory results obtained from preclinical studies on some radiolabeled nanoparticles, some of them have received FDA approval (Table 15) [248, 249]. Out of inorganic nanoparticles, Cornell dots (C dots), as silica nanoparticles, has been approved by FDA for targeted imaging in 2018 [249, 250]. Moreover, Vescan™ is the first commercialized liposomal radiotracer that was developed by Vestar Inc. as a liposome encapsulated with ¹¹¹In. This liposomal formulation has been extensively applied in cancer imaging and tumor diagnosis [14]. ^99mTc-albumin nanoparticles have also been commercialized as an imaging agent for lymphoscintigraphy and lymphatic mapping in inflammation, melanoma, and breast cancer via SPECT imaging. Nanocoll (EU) and Senti-Scint are trade names of albumin nanoparticles that could be used in SPECT imaging [251, 252].

Table 15 The FDA-approved radiolabeled nanoparticles

Conclusion

Nanomaterials have gained increasing attention during recent years due to their advantages in biomedical applications for in vivo delivery of diagnostic and therapeutic agents. Radiolabeled nanoparticles (radio-nanomaterial) are accurate, reliable, sensitive, and precise platforms for diagnostic and therapeutic applications. Nanoparticles can be labeled with appropriate radionuclides in different conditions. Regarding the approach adopted for radiolabeling of nanoparticles, they can be prepared in high yield and desirable radiochemical stability. Radiolabeling of nanomaterial is also an attractive way to assess their biodistribution and pharmacokinetics in the body with high accuracy. Radiolabeled nanoparticles present admirable platform for nuclear medicine imaging, including PET and SPECT imaging, in animals and humans for diagnosis of different diseases. There are different methods (direct and indirect) for radiolabeling various types of nanoparticles, and each radiolabeling technique has shown its own advantages and disadvantages. Among nanoparticles, the most radiolabeling procedures have been performed on different formulations of liposomes. Out of radiolabeled nanomaterials, the most studies on the targeted ones have been performed for gold- and protein-based nanoparticles as well. Of note is that cRGD peptide is considered as a common targeting agent for radiolabeled nanoparticles. The aim of this review was to elaborate the details on diagnostic radio-nanomaterial ever developed. There are still many paths that have not been explored in the application of diagnostic radionuclides in nanotechnology.