Nanoradiopharmaceuticals Based on Alpha Emitters: Recent Developments for Medical Applications

Abstract

The application of nanotechnology in nuclear medicine offers attractive therapeutic opportunities for the treatment of various diseases, including cancer. Indeed, nanoparticles-conjugated targeted alpha-particle therapy (TAT) would be ideal for localized cell killing due to high linear energy transfer and short ranges of alpha emitters. New approaches in radiolabeling are necessary because chemical radiolabeling techniques are rendered sub-optimal due to the presence of recoil energy generated by alpha decay, which causes chemical bonds to break. This review attempts to cover, in a concise fashion, various aspects of physics, radiobiology, and production of alpha emitters, as well as highlight the main problems they present, with possible new approaches to mitigate those problems. Special emphasis is placed on the strategies proposed for managing recoil energy. We will also provide an account of the recent studies in vitro and in vivo preclinical investigations of α-particle therapy delivered by various nanosystems from different materials, including inorganic nanoparticles, liposomes, and polymersomes, and some carbon-based systems are also summarized.

1. Introduction

Interest in theranostics and targeted therapies has fueled the advancement of new developments for beta and alpha radiation therapy in both the academic medical community and within the pharmaceutical industry. There has been significant recent interest in alpha particles due to their high cytotoxic potential that can be used effectively in cancer therapy. Targeted alpha-particle therapy (TAT) involves the irradiation of cancer cells, micrometastases, or tumors by emitting a single alpha particle or by a cascade of alpha particles within its vicinity.

Alpha particles ${\left({}_{}{}^4\mathrm{H}\mathrm{e}\right)}^{2+}$ are potent therapeutic effectors that have two advantages over beta therapies. First, the high linear energy (LET) of an alpha particle is two or three orders of magnitude greater than the LET of beta particles (100 keV/µm vs. 0.2 keV/µm). This unique characteristic of alpha particles allows a larger fraction of the total energy to be deposited in the cells, thus increasing therapeutic effectiveness. In sharp contrast, for the application of beta radiation, which has limited linear energy transfer, the cell must be hit by many thousands of beta particles before it is successfully killed [1,2,3,4,5]. In addition, the short-range of alpha radiation in human tissue of 50 to 100 µm (2–3 cell diameters), due to the high LET, compared to that of β particles (maximum ranges of 2 to 11 mm), allows the effective killing of targeted cancer cells due to the highly cytotoxic radiation, while limiting damage to surrounding healthy tissue. Therefore, targeted nanomedicine advances in alpha particles-based delivery offer the best potential for treating micrometastases while sparing normal tissues [6,7,8].

The high linear energy and the short-range features of alpha radiation are reflected in the production of DNA double-strand breaks (DSB), which increase the toxicity due to the limited ability to repair the damage, thus triggering instant cell death from a single atomic decay that passes through the nucleus. The toxicity to the tumor cells from the beta particles is less effective because of the onset of free radicals (indirect DNA damage), allowing a most efficient DNA reparation [9]. This increased biological efficacy of alpha particles is independent of oxygen concentration, dose rate, and the cell cycle stage. Moreover, 1–3 tracks through the nucleus can cause cell death [1,2,3,10,11,12,13,14,15].

Despite the effectiveness offered by the use of alpha particles in cancer therapy, some disadvantages, including limited availability or low production for medical applications, have impeded their widespread use in cancer therapy. Although there are more than 100 radionuclides that decay by alpha emission, most of them have either too short or too long half-lives for therapeutic use, or their chemical properties do not allow their optimal use in medicine. The choice of suitable radionuclide depends on multiple criteria: half-life, decay scheme, recoil energy, associated emission, physical and chemical properties, and availability.

Table 1. Physical properties of main alpha emitters for TAT.

Nuclide	t1/2	Main Emissions	Energy (MeV)	Energy Recoil (KeV)	Range (µm)	Daughters	t1/2	Decay	Energy (MeV)
227Th	18.7 d	α	6	---	50–70	223Ra
225Ac	10 d	α	5.8	---	50–90	221Fr	4.8 m	A	7
								γ	0.218
				105		217At	32.3 ms	A	7
				116		213Bi	45.6 m	A	6
								β−	0.444
								γ	0.440
				132		213Po	4.2 μs	A	8
						209Tl	2.2 m	β−	0.659
				160		209Pb	3.5 h	β−	0.198
						209Bi	Stable	---	---
223Ra	11.4 d	α	5.7	108.4	50–70	219Ra	3.96 s	A	6.8
				126		215Po	1.78 ms	A	7.4
				140		211Pb	36.1 m	β−	---
						211Bi	2.13 m	A	6.6
								β−	---
						211Po	0.516 s	A	---
				128		207Tl	4.77 m	β−	---
						207Pb	Stable	---	---
213Bi	45.6 m	α	6	50–90	132	213Po	4.2 μs	A	8
		β−	0.444			209Tl	2.2 m	β−	0.659
		γ	0.440			209Pb	3.5 h	β−	0.198
						209Bi	Stable	---	---
212Bi	60.6 m	α	6.1	40–100	---	212Po	0.3 µs	A	---
		β−				208Tl	3.1 m	β−	---
						208Pb	Stable	---	---
211At	7.2 h	α	5.9	55–80	116	207Bi	33.4 y	β−
		EC				211Po	0.516 s	A	---
						207Pb	Stable	---	---

shows the physical properties of several alpha emitters used in TAT [5]. The most difficult problem with the alpha particle management is associated with the radioisotope daughters release as well as lack of appropriate bifunctional chelating systems affording in vivo stability, a requirement to achieve selective targeting of alpha emitters at tumor sites [5,16]. Currently, targeted therapy using alpha-emitting isotopes presents an opportunity in the development of cancer therapeutic agents for treating various neoplastic diseases.

2. Alpha Emitters Used in TAT

Producing alpha emitters implies separating them from natural radionuclides with long half-lives and a combination of alpha emitters that can be generated from either: (a) legacy material, (b) nuclear reactors (neutron irradiation), or (c) accelerators (irradiation with charged particles). After their production, separating the desired radionuclide from the impurities is extremely important [5,16]. Several chemical separation methods, including precipitation, distillation, liquid-liquid extraction, ion exchange, or solid-phase extraction resin separations, can be used to produce radiopharmaceutical-grade alpha emitters for TAT applications [5].

2.1. Actinium-225 (²²⁵Ac)

Actinium is considered to be the most potent alpha emitter for use in TAT, since it decays to stable ²⁰⁹Bi (t_1/2 = 1.9 × 10¹⁹ y) through a series of six daughter radionuclides, with a total emission of four α particles with an energy of 5.8 MeV (total energy of 28 MeV) and associated tissue ranges of 50 to 90 µm (Figure 1) [3,13,17].

²²⁵Ac can be obtained either from the decay of ²³³U or by proton irradiation of ²²⁶Ra in a cyclotron through the reaction ²²⁶Ra(p,2n) ²²⁵Ac. However, the main source is its parent, ²²⁹Th (t_1/2 = 7932 y), which is the first decay product of ²³³U (t_1/2 = 1.592 × 10⁵ y) and is produced by radiochemical extraction [18]. Currently, there are three ²²⁹Th sources worldwide that can provide ²²⁵Ac: Institute for Transuranium Elements (ITU) in Karlsruhe, Germany; Oak Ridge National Laboratory (ORNL), USA; and Institute of Physics and Power Engineering (IPPE) in Obninsk, Russia. The overall production of ²²⁵Ac from the available ²²⁹Th sources is approximately 62 GBq per year [2,6].

Actinium isotopes are typically +3 ions with an ionic radius of 112 pm; they can be used as a parent radionuclide for the production of a ²²⁵Ac/²¹³Bi generator or used directly for the radiolabeling of targeting systems [5,19].

The main disadvantages of ²²⁵Ac are its low availability, recoil energy, and the complicated chemical properties of the daughters that decrease the stability of radiolabeled constructs and represent a risk for in vivo therapeutic applications [19].

2.2. Astatine-211 (²¹¹At)

Astatine (t_1/2 = 7.2 h) is a halogen and the only non-metal among the alpha emitters used for therapeutic applications [5]. This radionuclide decays through a branched pathway. The first branch decays to ²¹¹Po (t_1/2 = 526 ms), after which it decays to stable ²⁰⁷Pb. In the second branch, it decays to ²⁰⁷Bi (t_1/2 = 31.55 y), which then results in stable ²⁰⁷Pb after emission of X-rays. The decay of ²¹¹At is considered 100% alpha due to ²¹¹Po (the short-lived isotope) (Figure 2) [5,20]. ²¹¹A is produced in a cyclotron by α particles accelerated to energies of 28–29 MeV bombardment onto a metallic bismuth target via the nuclear reaction ²⁰⁹Bi(α,2n)²¹¹At, followed by simple dry distillation and isolation from irradiated bismuth target. This reaction generates energies between 21 and 40 MeV, with a maximum of about 31 MeV for medical applications [5,16]. An alternative method of ²¹¹At production is through a ²¹¹Rn/²¹¹At generator. The availability of ²¹¹At at present is limited due to the lack of appropriate cyclotrons. However, the new high-energy cyclotron, ARRONAX (Nantes, France), will contribute to a considerable increase in its production [2].

Astatine can exist in different oxidation states, having similar chemical and radiolabeling properties with heavy halogens such as iodine. The stability of the carbon-halogen bond decreases as we advance within this group in the periodic table, the carbon-astatine bond being the weakest. This bond is known to be sensitive to redox potential, especially in acidic conditions. The astatine release is closely related to the chelator atoms and their ability to stabilize it. In vivo destatination can cause accumulation in the thyroid, lung, stomach, or spleen [21].

Radiolabeling of non-activated aromatic compounds has an inherent disadvantage because such reactions are slow and result in low yields. However, the use of organometallic intermediates facilitates the reaction smoothly with high yields and good radiochemical purity [5,19].

²¹¹At has great potential to be used in TAT due to its low production cost. However, the main disadvantage is its low availability due to the lack of an accelerator capable of irradiating a target with an alpha-particle beam at 28 MeV [19].

2.3. Bismuth-213 (²¹³Bi)

Bismuth (t_1/2 = 45.6 m) is a mixed alpha/beta emitter that decays to metastable ²⁰⁹Bi as a decay product of the ²³³U series. Only 2.14% of ²¹³Bi decays via emission of an alpha particle to ²⁰⁹Tl with an average energy of 8.35 MeV, while 97.86% disintegrates via beta emission to the pure α emitter ²¹³Po (t_1/2 = 4.2 µs) (Figure 3). ²¹³Bi can be eluted from a ²²⁴Ra generator, owing to their short half-lives. However, the main drawbacks associated with short half-lives are that the entire processing of radiolabeling and quality control must be performed in a very short time [19]. Bismuth is the heaviest stable element in the periodic table with oxidation states of +3 or +5. Its coordination numbers vary from 3 to 9 or 10. Bi³⁺ forms stable complexes with electronegative atoms such as oxygen, nitrogen, sulfur, and halogens [5].

2.4. Lead-212 (²¹²Pb)

Lead is the father of ²¹²Bi and has been used for in vivo generated ²¹²Bi. The recoil energy of ²¹²Pb is 128 keV (Figure 3) [19].

2.5. Radium-223 (²²³Ra)

Radium (t_1/2 = 11.43 d) decays to stable ²⁰⁷Pb under emission of four α particles with energies between 5.7 and 7.4 MeV, releasing total energy of ~28 MeV, and is generated from the decay of ²²⁷Ac to ²²⁷Th by the emission of a beta particle followed by alpha emission to produce ²²³Ra (Figure 4) [16]. These radionuclides decay through a cascade of short-lived alpha- and beta-particle emitters to stable lead and bismuth, releasing high total energy of about 28 MeV [22].

Its production for clinical applications involves ²²⁷Ac and ²²⁷Th isolation from ²³¹Pa. However, the limited availability of the source of ²³¹Pa has recently led to the development of alternative production techniques based either on the isolation of the ²²⁷Ac by-product from the cyclotron production of ²²⁵Ac or after isolation of ²²⁷Ac obtained from actinium-227/beryllium-227 generator sources. Radium radionuclides too can be obtained from their long-living parent radionuclides: ²²³Ra from ²²⁷Ac (t_1/2 = 21.8 y), ²²⁴Ra from ²²⁸Th (t_1/2 = 1.9 y), and ²²⁵Ra from ²²⁹Th (t_1/2 = 7880 y) [2,19,22]. The main disadvantage of ²²³Ra, apart from their limited availability, is that the recoil energy and different chemical properties of the daughter isotopes decrease the stability of radiolabeled constructs [19].

2.6. Thorium-226 (²²⁶Th)

Thorium (t_1/2 = 30.7 m) is a promising radionuclide for use in targeted α emitter therapy due to the emission of four alpha particles with progressively decreasing half-life. ²²⁶Th can be eluted from generator systems containing the alpha emitter ²³⁰U (t_1/2 = 20.23 d) (Figure 5). Indeed, a successful cyclotron production of ²³⁰U has been demonstrated via proton irradiation of ²³¹Pa according to the reaction ²³¹Pa(p, 2n) ²³⁰U [2,5,23].

2.7. Terbium-149 (¹⁴⁹Tb)

Terbium (t_1/2 =  4.118 h) shows a branched decay where only 17% is disintegrated under the emission of an α particle to give rise to ¹⁴⁵Eu, while 83% decay to ¹⁴⁹Gd either via β⁺ emission (7%) or electron capture (76%). ¹⁴⁹Tb has two daughter nuclides, ¹⁴⁵Sm (t_1/2 = 340 d) and ¹⁴⁵Pm (t_1/2 = 17.7 y) (Figure 6).

The main production route for ¹⁴⁹Tb is light particle-induced reaction ¹⁵²Gd(p,4n) ¹⁴⁹Tb (Ep = 50 MeV) [5]. ¹⁴⁹Tb also has been produced at CERN in Geneva (Switzerland) by irradiation of a tantalum foil target with 1 or 1.4 GeV protons. The radionuclides that are generated during this spallation process are ¹⁴⁹Tb, ¹⁴⁹Dy, ¹⁴⁹Gd, and ¹⁴⁹Eu and are separated via cation exchange chromatography [2].

3. Alpha Emitters in Medical Applications

The high LET of alpha particles gives them an increased relative biological effectiveness (RBE), and therefore, they are capable of destroying tumors causing limited collateral damage to the surrounding healthy tissue. Nevertheless, the use of alpha particle-emitting radionuclides is hampered by the following issues: low availability and high costs, and are too short-lived (one hour or less), making transport of the radionuclides from the place of production to treatment sites highly challenging. Therefore, radionuclides with long half-lives such as ²²⁵Ac and ²²³Ra are used more frequently in clinics. It is important to recognize that the long-lived α emitters exhibit complex decay chains in which the daughter radionuclides receive high recoil energies (~100 keV) when the α particle is emitted. Therefore, alpha emitters require the use of vectors with high affinity and specificity in addition to maintaining suitable in vivo stability and rapid absorption by target cells. Several candidate isotopes for TAT are currently under preclinical and clinical evaluation, including ²²⁵Ac, ¹⁴⁹Tb, ²¹¹At, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, and ²²⁴Ra [7,24].

4. Recoil Energy

Alpha particle use is mainly restricted by recoil energy despite their well-known large cytotoxic effects. The recoil energy causes the breakdown between the targeting molecule or chelate and the radionuclide. This release implies the emission of the first alpha particle and the parent transformation into a new daughter owning different chemistry (Figure 7). Subsequently, there is a recoil of the main atom away from the chelate. Part of the decay energy is transferred to a daughter nucleus due to the momentum conservation law, and subsequent intermediates that emit alpha (daughter) particles have recoil energy due to the daughter nuclide of approximately 100 keV. This has the ability to break at least 10,000 chemical bonds (assuming 10 eV/one bond). This is undesirable because alpha decay will cause bond breakage and the escape of daughter atoms that can target and irradiate healthy organs and tissues, thus reducing the therapeutic dose administered to the diseased site [11,19,23,25,26,27].

The distribution and distance covered by the recoiling daughters through the body depend on the recoil energy, diffusion processes, blood flow, and the location where the recoil atoms are released. Although most of the time, the recoils will be released in the bloodstream, and their fate will be determined by their affinity to organs and tissues provided that they live long enough to reach their biological destination [23,28]. Another problem associated with working on alpha particles is that they are emitted isotropically, and the emission directions of the successive decays in the decay chains are not correlated [23]. For these reasons, parameters such as the biological half-life of the radionuclide and that of its radioactive daughters, the in vivo stability of the carrier, uptake in the reticulum endothelial system (RES), plasma clearance, elimination routes, released activity, the fraction of the radioactive daughter nuclei, and the metabolic fate play an important role and should be taken into account for the in vivo evaluation of the radiopharmaceuticals used for TAT [27].

5. Strategies

Alpha particles are extremely effective in killing tumor cells, but they can also cause measurable damage to healthy tissues due to the chemical instability of radiolabeling. Therefore, it is of profound importance to achieve the optimum in vivo stability of the radiopharmaceutical(s) while confining alpha emitters to the tumor microenvironment. To meet these stringent needs, strategies are being developed to promote retention of the decay daughters and reduce recoil energy impact. We discuss here three important strategies: (1) encapsulation of radionuclides in a nanocarrier, (2) use of targeting vehicles (antibodies or peptides), and (3) local administration.

5.1. Encapsulation of Radionuclides in a Nanocarrier

This strategy, along with targeted design, facilitates high target accumulation, mainly for radionuclides that present branched decay such as found in ²²⁵Ac, ²²³Ra, and ²²⁷Th. This strategy allows encapsulation of radionuclides within a nanocarrier (for example, liposomes, nanoparticles, etc.) that must be large enough to sequester physically all the setbacks of the decay chain in its structure and thus prevent its release and that of the daughter radionuclides escaping from the target site, all aimed at preventing nonspecific radiotoxicity [7,9,11,13,23,24].

5.2. Targeting Vehicle

This option consists of developing targeting vehicle molecules such as antibodies or receptor-specific peptides with high-selectivity targeting vectors to facilitate rapid and effective internalization in tumor cells while limiting irradiation of non-target tissues. This strategy has shown clinical potential through both antibodies (pre-targeted radioimmunotherapy (PRIT)) and peptides (peptide receptor radionuclide therapy (PRRT)) [10,20,24,28].

5.3. Local Administration

Local administration of alpha emitters allows the deposition of radioactivity directly into the target site via injection. It is prudent that the alpha-emitting systems be applied in a low irrigation region to ensure that no daughter radionuclides may infiltrate blood circulation [21,28].

In this review, we provide elaborate details on the first strategy wherein mother radionuclides are encapsulated into a nanoparticle to confine all recoils of their decay chain within its structure. Such a nanoparticle requires a suitable size and structure and should allow an adequate surface functionalization to enable efficient targeting. The success of this approach hinges heavily on the chemical nature and dimensions of nanocarriers. The recoil energies of the daughter nuclides and their displacement range within the material that the nanocarrier is made up of needs to be carefully chosen because the material and size of the nanoconstruct would determine the energy loss of recoils in nanoconstruct materials [16].

There are three important factors to consider when designing a nanocarrier. First, the distribution of daughter recoils is mitigated over time, so their distribution within the organism would also depend on their half-life. Second, daughter recoils distribution is mitigated by size and the chemical constitution of the material of nanocarrier due to the recoiling nuclide consuming some of its energy as it passes through the nanocarrier. Finally, recoils distribution mitigation can be handled by the number of nanocarriers or layers because, even though the recoil ion may escape a nanocarrier or layer, the probability of it entering or slowing down within surrounding nanoconstructs is relatively high [9,27,29,30,31]. In addition, for every subsequent radioactive daughter, the probability of remaining within the physical limits of the liposomal carrier is decreased as compared to the corresponding previous radioactive daughter.

6. Chelating Agents

The high energy of α particles and their superior cell killing power demands high stability with the targeting vector, as well as high specificity, good in vivo stability, and fast uptake by target cells. Very long times in circulation, even longer than the life of radionuclides in media, can cause irradiation and nonspecific off-target toxicity [5,9]. It is well known that the main limitation for the application of α emitters in radionuclide therapy is the escape of daughter radionuclides of radiopharmaceuticals since chelating agents (cyclic or linear) are not capable of withstanding the recoil energy of decay daughters (100–200 keV), which is greater than the energy of any chemical bond [16].

In the bonding of a radionuclide(s) with targeting vector(s), the conjugation of such vector(s) with “bifunctional” chelating agents (BCA) is involved. The most common bioconjugation techniques involve peptide coupling reaction between a carboxylic acid and a primary amine, the peptide coupling between activated esters of tetrafluorophenyl (TFP) or N-hydroxysuccinimide (NHS), and a primary amine; a thiourea bond formation between an isothiocyanate and a primary amine; a thioether bond formation between a maleimide and thiol; the standard Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition (“click” reaction) between an azide and an alkyne; and Diels–Alder “click” reaction between a tetrazine and transcyclooctene [32]. BCA, generally, possesses two functional groups, one is a metal-binding moiety (e.g., DTPA or DOTA), and the other is a chemically reactive functional group, capable of binding to a targeting moiety such as peptides or antibodies [19,22,33,34,35]. The structure and physical properties of the radiometal-chelate complex have a great impact on the pharmacokinetic properties of a radiopharmaceutical. Numerous investigations suggest that changing the chelating agent considerably modifies the biodistributions and in vivo stability [32].

Acyclic ligands are more flexible, offering faster chelation kinetics, but they afford lower in vivo stability. Macrocyclic ligands offer rigid structures with facile chelate formations that have higher stability in vivo but with slower kinetics. The ability to quantitatively radiolabel in less time at room temperature is always an attractive property of acyclic chelators, whereas macrocycles often require heating for extended times. Fast room temperature radiolabeling becomes a need when working with bifunctional chelating agents conjugates with heat-sensitive molecules (e.g., antibodies, peptides) or when working with short half-life radionuclides (e.g., ^212/213Bi) [32].

A description of the most common bifunctional reagents used with alpha-emitting radionuclides is provided in the following sections, and their structures are depicted in

Table 2. Structures of acyclic and macrocyclic common bifunctional reagents used with alpha-emitting radionuclides.

DOTA and Bifunctional Derivatives		Mainly Alpha Radionuclides	Ref
DOTA: 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid			[32,36,37,38,39,40,41,42,43]
DOTAGA-anhydride		212Pb2+, 225Ac3+, 212/213Bi3+, 223Ra2+, 149Tb, 226/227Th
DOTA-NHS-ester
DOTAGA, R = amide, DOTA-NHS-ester
p-SCN-Bn-DOTA (C-DOTA)
DOTA derivatives TCMC, 3p-C-DEPA, and bifunctional derivatives
3p-C-DEPA: 2-[(carboxymethyl)]-[5-(4-nitrophenyl-1-[4,7,10-tris-(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)-amino]acetic acid			[32,44,45,46,47]
3p-C-DEPA-NCS
TCMC, 1,4,7,10-tetrakis(carbamoylmethyl)- l,4,7,10-tetraazacyclododecane		212Pb2+,212/213Bi3+
p-SCN-Bn-TCMC
NOTA, NETA, TACN-TM, and bifunctional derivatives
NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid			[32,43,48]
p-SCN-Bn-NOTA (C-NOTA)
NETA: {4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid
C-NE3TA-NCS		212/213Bi3+, 212Pb2+
3p-C-NETA
C-NETA-NCS
DTPA, bifunctional derivatives, and others
DTPA: diethylenetriaminepentaacetic acid			[5,32,36,41,42,49,50]
p-SCN-Bn-CHX-A″-DTPA
p-SCN-Bn-1B-DTPA
CHX-A”-DTPA, 2-(p-isothiocyanatobenzyl)- cyclohexyldiethylenetriaminepentaacetic acid		212/213Bi3+, 225Ac3+, 223Ra2+, 149Tb, 226/227Th
p-SCN-Bn-1B4M-DTPA
(Me-2,3-HOPO)4-Bn-NCS		226/227Th
HEHA, PEPA, and bifunctional derivatives
HEHA: 1,4,7,10,13,16-hexaazacyclo-hexadecane-N′,N″,N‴,N,N⁗,N″″′-hexaacetic acid			[32,36,37,38]
p-SCN-Bn-HEHA (C-HEHA)
PEPA: 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N‴,N⁗-pentaacetic acid
p-SCN-Bn-PEPA (C-PEPA)
MACROPA		225Ac3+

.

6.1. ²²⁵Ac

Larger ligands, HEHA and macropa, have been explored for ²²⁵Ac for high in vitro and in vivo stability conjugates obtained at room temperature [36,37,38]. Chappell et al. (2000) developed 2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA-NCS) as a chelating agent and radiolabeled three mAbs with ²²⁵Ac. In this study, ²²⁵Ac-HEHA showed unstable coordination chemistry and release of the daughter radionuclides in vivo [37]. The HEHA and PEPA macrocycles have been widely used with ²²⁵Ac, but recently it has been shown that DOTA and derivates have better properties in vivo; however, the high temperatures required for radiolabeling of ²²⁵Ac with DOTA are very unfavorable for sensitive antibodies [32]. The current method used for synthesizing ²²⁵Ac-DOTA antibody conjugates employs two steps: first, radiolabeling the p-SCN-Bn-DOTA (BFC) at 60 °C with ²²⁵Ac; second, the antibody conjugation (thiourea coupling); and finally, the purification steps prior to in vivo utility [37,39,40]. Song et al. (2009) compared the therapeutic effects of ²²⁵Ac, ²¹³Bi, and ⁹⁰Y using an antibody vector and demonstrated ²²⁵Ac to be the most effective in terms of in vivo characteristics. On the other hand, biodistribution studies of ²²¹Fr and ²¹³Bi (²²⁵Ac daughters) revealed accumulation in kidneys and contributed to the long-term renal toxicity [41]. Clinical trials in humans with ²²⁵Ac-DOTA-HuM195 (humanized anti-CD33 monoclonal antibody) have shown DOTA being the current “gold standard” for ²²⁵Ac [39,49].

6.2. ²¹²Pb

DOTA has been used successfully with ²¹²Pb; however, slow radiolabeling and low stability were not ideal. Today, the chelator TCMC produced by replacement of the carboxylic acid donor arms of DOTA with amide arms is considered the best chelator available for ²¹²Pb [42,44,45].

6.3. ^212/213Bi

The short half-life of ²¹²Bi requires rapid radiolabeling kinetics, thus requiring the use of acyclic ligands. Several studies have been reported on the use of derivatives of functionalized diethylenetriamine pentaacetic acid (DTPA) conjugated to mAbs and peptides, including the cyclic DTPA anhydride derivative, 2-(p-isothiocyanatobenzyl)-DTPA (SCN-Bz-DTPA), isothiocyanatobenzyl derivative of C-functionalized trans-cyclohexyl DTPA (CHX-A″-DTPA), and 2-(p-isothiocyanatobenzyl)-6-methyl-DTPA (Mx-DTPA, 1B4M-DTPA). DTPA complexes have shown good stability in vitro, but only the CHX-A″-DTPA forms suitably stable complexes in vivo. DOTA has also been successfully used for labeling mAbs with ²¹²Bi that demonstrated in vivo stability in an animal model. However, the slow kinetics of radiolabeling is considered a disadvantage [5,36].

In a comparative study of various macrocyclic ligands (e.g., NOTA, TETA, and DOTA), DOTA was found to be the most promising ligand since it formed stable complexes with both lead and bismuth [43]. Later, a derivative of DOTA was studied in which the pendant carboxylate arms were replaced by carbamoyl methyl arms, and the macrocyl TCMC or DOTAM emerged. The antibody system with the new macrocyl TCMC had an easier synthesis process than that of DOTA and showed a high radiochemical yield (>95%); in addition, they showed a loss of between 15% and 30% of ²¹²Bi from the system [46].

Chong et al. (2008) evaluated the NETA and bifunctional derivatives C-NETA and C-NE3TA. Their results showed good stability with ^212/213Bi during 11 days in blood serum. Biodistribution studies of C-NETA complexes demonstrated excellent clearance and good stability in vivo [48]. Later, in 2011, Chong et al. showed 3p-C-NETA and trastuzumab immunoconjugates exhibit rapid radiolabeling kinetics and excellent in vitro and in vivo stability [51]. Song et al. (2011) managed to improve the radiolabeling kinetics of ^212/213Bi with 3p-C-DEPA (derived from DOTA) compared to DOTA, without observing significant changes in in vivo stability. 3p-C-DEPA is today considered to be the “gold standard” for this radionuclide [47].

6.4. ²²³Ra

Radiolabeling for ²²³Ra has been developed with DTPA, DOTA, and calix [4] arene tetraacetic acid chelating systems. It has been shown that calix [4] arene provides the most stable complexes, but its stability is not optimal for in vivo use. So far, a vast majority of the ligands used have failed in the development of stable in vivo chelates of ²²³Ra. This is where the application of nanotechnology appears to circumvent this vexing problem. Radiolabeling of ²²³Ra through encapsulating it in different types of nanomaterials is an emerging approach to achieving in vivo stable nanoradiopharmaceuticals of ²²³Ra [5,51,52,53].

6.5. ¹⁴⁹Tb

Non-radioactive terbium has shown high stability when chelated to DOTA, DOTA derivatives, and CHX-A″-DTPA [5]. Beyer et al. (2012) demonstrated in vitro studies of ¹⁴⁹Tb chelated to conjugated MAb-CHX-A″-DTPA [50]. Recently, in vivo study using four Tb isotopes attached to the same conjugate cm09 (DOTA ligand attached targeting vector) demonstrated their potential for in vivo applications [54]. These results demonstrated that terbium radioisotopes can be readily chelated using CHX-A″-DTPA and DOTA [52].

6.6. ^226/227Th

The antibody labeling with ²²⁷Th has been carried out using mainly the DOTA chelating agent. The antibody labeling with ²²⁷Th by a two-step process resulted in 6–7% yields [55]. CHX-A″-DTPA (DTPA derivatives) may be useful for ²²⁶Th, and DOTA-Bn-NCS has been used for labeling of mAbs with ²²⁷Th, but the labeling yields were low [52]. Ramdahl et al. (2016) presented an octadentate bifunctional chelator containing hydroxypyridinone (HOPO) moieties. (Me-3,2-HOPO)₄-Bn-NCS facilitates the labeling of mAbs with ²²⁷Th in 30 min and at room temperature, producing high radiochemical yields (>96%). Their results showed that (Me-3,2-HOPO)₄-chelated ²²⁷Th has good in vivo stability [56].

6.7. ²¹¹At

Labeling of ²¹¹At with conjugates based on phenyl rings has shown in vivo instability and low labeling yields. Recently, the formation of aromatic boron-astate bonds has been proposed as an attractive alternative to achieve optimum in vivo stability. The advantage of this procedure is that the conjugation to protein and/or antibodies can be performed in the first step, thus reducing the reaction time [5]. Wilbur et al. (2012), in their studies using the closo-decaborate²⁻ moiety (aromatic boron cage moieties), developed immuno-conjugates that can be rapidly labeled (under 2 min) with high radiochemical labeling yields (80–95%) with good in vivo stability [57].

7. Targeting Vectors

The successful implementation of α emitters in medicine requires their effective targeting with high selectivity at tumor sites. Targeting vectors, including mAbs, peptides, or nanoparticles (e.g., liposomes, micelles, dendrimers), will play prominent roles in the development of targeted α-emitting radiopharmaceuticals.

There are two main approaches by which a targeting vector can be linked to an α-emitting nuclide. In the first approach, the bifunctional reagent is labeled with an α-emitting nuclide with subsequent conjugation with the tumor-targeting vector. In the other alternative, the bifunctional reagent is first conjugated to the targeting vector, and then the conjugated vector is radiolabeled with an alpha emitter [5]. Using ²¹¹At, conjugations with vectors such as MAb have been reported [19,58]. In the first clinical trial (phase I/II study), a ²¹¹At-labeled MAb (²¹¹At-ch81C6) was evaluated to treat brain cancer. In the second clinical trial (phase I study), ²¹¹At-labeled MAb (²¹¹At-MX35F(ab′)₂) was administered in ovarian cancer patients. Both studies showed that the toxicity was not significant [59,60]. A ²¹¹At-labeled anti-CD45 conjugate (²¹¹At-BC8-B10) is being evaluated in phases I/II clinical trials (clinicaltrials.gov NCT03128034 and NCT03670966). ²¹¹At-BC8-B10 has been administered intravenously to treat patients with acute myeloid or lymphoblastic leukemia and myelodysplastic syndrome [61].

The in vivo generator ²¹²Pb/²¹²Bi has been evaluated in radiotherapy directed at the human epithermal growth factor receptor (HER2 or HER1). Preclinical studies of trastuzumab conjugated with the bifunctional agent TCMC-Bn-NCS have been carried out for radiolabeling of ²¹²Pb. Preclinical studies of trastuzumab conjugated with the bifunctional agent TCMC-Bn-NCS have been carried out for radiolabeling of ²¹²Pb (²¹²Pb-TCMC-trastuzumab) evaluating the system alone or in combination with drugs such as paclitaxel, gemcitabine, etc. for treatment of ovarian, prostate, and pancreatic cancer [62,63]. TCMC-Bn-NCS have been conjugated with different antibodies. In 2016, Milenic et al. evaluated the biodistribution, pharmacokinetics, and toxicity of ²¹²Pb-TCMC-trastuzumab in a phase I clinical trial in patients with HER2-expressing ovarian cancer. The results showed that the redistribution of radionuclide was minimal and the myelosuppression was not significant [63], Later, in 2018, Milenic et al. evaluated the conjugation of panitumumab F(ab′)₂ with TCMC as a targeting agent for epidermal growth factor receptor [64].

In addition, preclinical and clinical trials with different monoclonal antibodies (mAbs) and peptides labeled with ²¹³Bi for treating leukemia (²¹³Bi-HuM195), lymphoma (²¹³Bi-anti-CD20 MAb), melanoma (²¹³Bi-9.2.27), bladder cancer (²¹³Bi-anti-EGFR MAb), glioblastoma (²¹³Bi-substance P), and neuroendocrine tumors (²¹³Bi-DOTATOC) have been reported [64,65,66,67,68,69], and ²²⁵Ac-labeled mAbs and peptides for acute myeloid leukemia (²²⁵Ac-DOTA-NCS with lintuzumab or ²²⁵Ac-HuM195mAb), for prostate cancer (²²⁵Ac-PSMA-617), glioma (²²⁵Ac-Substance P), and neuroendocrine tumors (²²⁵Ac-DOTATOC) have been evaluated [13,18]. In clinical trials, hundreds of patients with prostate cancer have undergone the treatment with ²²⁵Ac and ²¹³Bi-labeled PSMA-617 and have presented excellent responses [67,70].

Hagemann et al. in 2016 evaluated ²²⁷Th-labeled anti-CD33 antibodies for the treatment of acute myeloid leukemia in vitro and in vivo [71] and later in 2017 evaluated ²²⁷Th-labeled anti-CD70 to immunotherapy [72]. Two clinical trials (clinicaltrials.gov NCT02581878 and clinicaltrials.gov NCT03507452) have been reported. The first was to evaluate an anti-CD22 MAb conjugate, ²²⁷Th-epratuzumab (BAY1862864), to establish the dose in patients with CD22 positive non-Hodgkin’s lymphoma (NHL), and in the second trial, a ²²⁷Th-labeled MAb conjugate (BAY2287411) was used to evaluate the safety and maximum dose in patients with epithelioid malignant mesothelioma and ovarian cancer [5].

8. Nanoradiopharmaceuticals Based on Alpha Emitters

In the continued quest and exploration of nanoparticles as drug carriers, several approaches have been developed to load nanoparticles with α-emitting radionuclides to dissipate the recoil energies of the daughter radionuclides in the nanoparticles and to avoid their release. Thus far, zeolites, liposomes, polymersomes, and metal-based particles have been studied to retain the recoil energy of alpha-emitting radionuclides and are presented in Table 3.

Nanoparticle-based systems have been designed to improve biodistribution, stability, specificity, pharmacological and targeting properties, and daughter retention, as well as to exploit the theranostic potential for dual imaging and therapy.

Inorganic nanoparticles (NPs) made of lanthanide phosphate, zeolites, magnetite, hydroxyapatite, barium sulfate, and calcium carbonate have shown high encapsulation of alpha emitters, while decay daughters have been partially retained. Kucka et al., in 2006, prepared ²¹¹At-radiolabeled silver nanoparticles and coated the surface with poly(ethylene oxide) polymer. The radiolabeling yields were higher than 95%, but the extent of retention in tumors was not reported [73]. In 2011, Woodward et al. developed inorganic lanthanum phosphate nanoparticles to incorporate ²²⁵Ac [²²⁵Ac(La(²²⁵Ac)PO₄)] conjugated with the monoclonal antibody mAb201B. Results showed almost complete retention of ²²⁵Ac into nanoparticles; however, 50% of the daughter nuclides (²²¹Fr and ²¹³Bi) were released in in vitro investigations. In vivo release of ²¹³Bi in lungs, liver, spleen, and kidney, indicating some release of this daughter nuclide following nuclear recoil, was observed despite its encapsulation in the nanoparticle [74]. Rojas et al. have investigated core + 1 shell and core + 2 shells of LaPO₄ nanoparticles (NPs with two layers of cold LaPO₄ deposited on the core surfaces) containing either ²²³Ra or ²²⁵Ra/²²⁵Ac to evaluate their ability to retain the parent isotopes and associated daughter products in vitro. They showed that the addition of cold LaPO₄ shells to the NPs surfaces significantly improves the retention of the daughters’ nuclides. The core + 1 shell retained 88% of ²²³Ra over 35 days. However, in the core + 2 shells NPs, the retention of ²²³Ra and its daughter (²¹¹Pb) was improved to 99.9% over 27 days. Additionally, the retention of ²²⁵Ra/²²⁵Ac parents was approximately 99.9% and 80% for the ²²¹Fr and ²¹³Bi daughters, respectively, over 35 days [75]. In 2013, Piotrowska et al. synthesized NaA nanozeolite (30–70 nm), in which the percentage of absorption for ²²⁴Ra and ²²⁵Ra was above 99.9%. ²²⁴Ra-nanozeolite showed high stability in physiological and PBS solutions for up to 24 h, while ²²⁵Ra-nanozeolite was stable up to 2 days. After this time, the leakage of ^224,225Ra from the nanozeolites was below 0.5%. In contrast, labeled nanozeolites were unstable in EDTA and cysteine solutions [22]. Nanozeolite carriers were also functionalized with silane-PEG-SP and labeled with ²²³Ra. The synthesis rendered 50–80 nm nanoparticles. The leakage of ²²³Ra was less than 0.5%, and the release of ²¹¹Pb and ²¹¹Bi (decay products) was 2–5% in human serum, which corresponds to 90% to 95% retention of the decay products [76]. McLaughlin et al. demonstrated that multilayered nanoparticles (NPs) {La_0.5Gd_0.5}PO₄@GdPO₄@Au doped with ²²⁵Ac (diameter 27 nm) managed to retain 99.99% of the ²²⁵Ac within 3 weeks and 88% of the decay products (²²¹Fr and ²¹³Bi) in studies in vitro. The in vivo behavior showed that ²²⁵Ac was predominantly present in the liver and spleen, while retention of ²¹³Bi (the breakdown of ²²⁵Ac in NPs) in the liver and spleen after 24 h was 84–63% [31]. Table 3.

Table 3. Summary of encapsulation of alpha emitters experiments reported in the literature.

Radionuclide	Nanoparticle (Type)	Retention/Release of Daughter Nuclides	Labeling Yield	Size	Ref
225Ac	225Ac(La(225Ac)PO4)	Retention ~100% of 225Ac Retention ~80% of 213Bi (daughter of 225Ac decay) Retention ~50% of the daughters (221Fr and 213Bi; 1 month) Release 50% of the daughter nuclides 221Fr and 213Bi in vitro	66%	3–5 nm	[74]
	Multilayered {La0.5Gd0.5}PO4@GdPO4@Au doped with 225Ac	Retention ~100% of 225Ac (3 weeks) Retention of 88% 221Fr and 213Bi (decay products) Retention of 84–63% of 213Bi (breakdown of 225Ac in NPs) in liver and spleen after 24 h in vivo	~76%	27 nm	[31]
	Multilayer {La0.5Gd0.5}(225Ac)PO4@4GdPO4 shell@Au	Retention >99.9% 225Ac (3 weeks) Retention in vivo of 213Bi (daughter of 225Ac decay) in lung tissue > 70% (1 h) and approximately 90% (24 h) 221Fr retention ~60% (3 weeks)	---	---	[77]
	Multilayer {Gd0.75La0.25}(225Ac)PO4@4 LaPO4 shell@Au	Retention ~100% of 225Ac (3 weeks) Retention of 221Fr 60–89%	76%	19.9 ± 6.5 nm	[78]
	Gadolinium vanadate (GdVO4)	Leakage of 221Fr (1st daughter of 225Ac) and 213Bi (3rd daughter of 225Ac) was 55.4 ± 3.6%, and 25.6 ± 1.5%, respectively (5 days) Leakage of 221Fr and 213Bi was of 41.1 ± 2.5% and 19.8 ± 1.16%, respectively (11 days) Leakage of 223Ra 73.0 ± 4.0% (4 days), and 30.0 ± 1.7% (33 days) The leakage of 211Pb was 44.8 ± 3.1% (35 days)	---	---	[24]
	Gadolinium vanadate core (Gd(225Ac)VO4) and core + 2 shell (Gd(225Ac)VO4/2GdVO4)	Leakage of 225Ac was 15% and 2.4% from core and core + 2 shells Leakage of 227Th was 3% and 1.5%, from core and core + 2 shells, respectively Leakage of 221Fr (first decay daughter) from core was 40.6 ± 2.4% (2 days) and ~69.5 ± 4.9% (23 days) Leakage for 213Bi from core and core + 2 shell was 22.5 ± 1.3% and 19.6 ± 1.9%, respectively Leakage of 227Th was ~3% (15 days) and 1.6 ± 0.3% (18 days) Leakage of 223Ra from core was 10.0 ± 0.7% (1 day) and 39.0 ± 2.2% (2 weeks)	Yield of 225Ac was 77.1 ± 13.2% and 96.6 ± 1.6% for core and core + 2 shell, respectively Yield of 227Th was 62.9 ± 2.7% and 81.9 ± 1.2% for core and core + 2 shell, respectively	3.6 ± 0.9 nm for core and 4.4 ± 1.0 nm for core + 2 shell	[79]
	Gd0.8Eu0.2VO4 core and core + 2 shells	The leakage of 221Fr from core was 40.9 ± 0.3% (2 days), 55 ± 3.6% (10 days) and 67.6 ± 3.3% (28 days) Leakage of 221Fr from core + 2 shells was 36.3 ± 6.2% (10 days) and 45.5 ± 3.6% (28 days) The leakage of 213Bi was <15% for core and ~22% for core + 2 shells	Yield core was 41.1 ± 16.5%, shells increased the yield up to 55%	6.1 ± 1.4 nm for core and 12.4 ± 2.0 nm for core + 2 shells	[80]
	Liposomes: Pegylated liposomes membrane charge (zwitterionic and cationic)	213Bi retention was 10% (650 nm) 225Ac retention in the zwitterionic liposome was ~88% (30 days) 225Ac retention in cationic liposomes was 54%	6.4–10.0%	200/400/650 nm	[11]
	Liposomes	225Ac retention of 81 ± 7%	55–73%	121 ± 6 nm	[81]
	Liposomes	Retention of 225Ac with up to three 225Ac nuclides per every 2 liposomes	58.0–85.6%	107 ± 2 nm	[82]
	Multivesicular liposomes (MUVELs)	Retained 98% of 225Ac (30 days) Retention of 213Bi was 31% (758 nm)	---	---	[83]
	Polymersomes	Retention 221Fr was 71.5% (800 nm, 24 h) Retention 213Bi was 74% (800 nm, 24 h)	213Bi was 83 ± 75% (100 nm) 225Ac was 67 ± 0.8% (100 nm)	100, 200, 400, and 800 nm	[28]
	Double-layered polymersomes	Retention of ~100% of 221Fr (800 nm) Retention of 213Bi (recoil atoms) was 80% (800 nm)	---	300–800 nm	[26]
	Polymersomes	Retention of 225Ac was 92 ± 3% Retention of 221Fr (first daughter) was ~20% Retention 213Bi was ~10%	89 ± 0.6%	100/200/400/800 nm	[84]
	Polymerosomes	---	>90% for 111In and >64% for 225Ac	---	[85]
	Polymerosomes	221Fr and 213Bi were retained at the tumor site 88 ± 9% and 89 ± 2%, respectively (2 days p.i.)	54–59%	97 ± 37 nm	[86]
	Fullerenes: 225Ac metallofullerene	---	---	---	[87]
	TiO2	Retained >95% of 225Ac and 221Fr (10 days)	99.8 ± 2.1%	25 nm	[88]
	Carbon nanotubes	Clear rapidly and shown to be therapeutically effective in vivo	---	---	[89]
	Carbon nanotubes	---	~95%	---	[90]
	Lipid vehicle	225Ac were retained to significant extents by during 6 h	62.7 ± 14.6%	106 ± 4 nm	[91]
223Ra	LnPO4 core and core + 2 shells NPs (Ln = La, Gd)	The core LaPO4 NPs retained up to 88% of 223Ra (35 days) Retention of 99.9% of 223Ra and 211Pb (daughter of 223Ra) the core + 2 shells (27 days) Retention ~100% of 211Pb (decay daughter) Retention of 99.98% of 225Ra/225Ac parents and ~80% for the 221Fr and 213Bi daughters in the core + 2 shells (35 days)	91% for LaPO4 core + 2 shell	3.4 nm for core and 6.3 nm for core + 2 shells	[75]
	Hydroxyapatite (HA)	No significant release of activity was detected	>95%	15 nm	[92]
	Hydroxyapatite (nHAp) and titanium dioxide (nTiO2)	The sorption efficiency on nHAp was 95 ± 5% and approximately 100% in the case of nTiO2	~95%	---	[93]
	Hydroxyapatite (HAP)	---	98%	Width up to 100 nm and length up to 500 nm	[94]
	Hydroxyapatite (HAP)	---	98%	900–1000 μm	[95]
	Liposomes	223Ra retention was 96 ± 1% (24 h) and 93 ± 2% (100 h) 228Ac retention was 95 ± 2% (24 h)	223Ra was 78 ± 6% 228Ac was 61 ± 8%	---	[96]
	Liposomes: Pegylated liposomal doxorubicin (PLD)	Liposomal 223Ra was relatively stable in vivo and was largely retained in liposomes	51–67%	80 nm	[97]
	223/224/225Ra-nanozeolite (NaA)	The leakage in PBS solutions of 224,225Ra-nanozeolites was below 0.5%	99.9%	30–70 nm	[22]
	223Ra-labeled nanozeolite	The leakage of 223Ra was <0.5% Release of 211Pb and 211Bi (decay products) was 2% (1 day) and 5% (6 days) in human serum Retention of 90–95% of 211Pb and 211Bi (decay products)	99.9%	50–80 nm	[76]
	BaSO4	---	20%	140 nm	[98]
	Superparamagnetic iron oxide nanoparticles Fe3O4 SPIONS	PBS, bovine plasma, and serum showed a cumulative release of 223Ra below 5%	85–99% (PBS)	4–26 nm	[99]
	Reduced graphite oxide	Desorption in PBS + BSA solution <5% of 99mTc Desorption in BSA in PBS <10% of 99mTc Desorption in PBS + BSA solution in 1 h raised to 35% for 90Y	Sorption was 10% for 223Ra, 90% of 99mTc, 80–100% for 207Bi and 90Y	4–6 nm	[100]
	Polyoxopalladates (Pd-POM): [224Ra]Na-a(Ra)Pd15	224Ra was incorporated into [224Ra] Na-Ba(Ra)Pd15, but the insertion is not selective (lead and bismuth Pd-POM can also be generated)	---	---	[101]
	Calcium carbonate microparticles	Retention >95% for 224Ra and its daughter nuclide 212Pb (1 week)	>80% of 224Ra and 212Pb (daughter nuclide)	---	[102]
211At	Silver core coated by PEO shell	---	50–97%	18.3–34.7 nm	[73]
	Ultrashort nanotubes	Retention was 19.6% and 11.0% for water and methanol, respectively	77.7–91.3%	20–50 nm in length and 1 nm diameter	[103]
	Gold nanoparticles (AuNPs)	---	>99%	5 and 15 nm	[104]
	Gold nanoparticles (AuNPs)	---	>99%	5 nm	[105]
212Pb	Liposomes	---	75%	---	[106]
	Liposomes	212Pb and 212Bi were 95% retained (20 h)	90 ± 2%	---	[107]
	Hydroxyapatite (HAP)	---	---	---	[108]

Table 3. Summary of encapsulation of alpha emitters experiments reported in the literature.

Radionuclide	Nanoparticle (Type)	Retention/Release of Daughter Nuclides	Labeling Yield	Size	Ref
225Ac	225Ac(La(225Ac)PO4)	Retention ~100% of 225Ac Retention ~80% of 213Bi (daughter of 225Ac decay) Retention ~50% of the daughters (221Fr and 213Bi; 1 month) Release 50% of the daughter nuclides 221Fr and 213Bi in vitro	66%	3–5 nm	[74]
	Multilayered {La0.5Gd0.5}PO4@GdPO4@Au doped with 225Ac	Retention ~100% of 225Ac (3 weeks) Retention of 88% 221Fr and 213Bi (decay products) Retention of 84–63% of 213Bi (breakdown of 225Ac in NPs) in liver and spleen after 24 h in vivo	~76%	27 nm	[31]
	Multilayer {La0.5Gd0.5}(225Ac)PO4@4GdPO4 shell@Au	Retention >99.9% 225Ac (3 weeks) Retention in vivo of 213Bi (daughter of 225Ac decay) in lung tissue > 70% (1 h) and approximately 90% (24 h) 221Fr retention ~60% (3 weeks)	---	---	[77]
	Multilayer {Gd0.75La0.25}(225Ac)PO4@4 LaPO4 shell@Au	Retention ~100% of 225Ac (3 weeks) Retention of 221Fr 60–89%	76%	19.9 ± 6.5 nm	[78]
	Gadolinium vanadate (GdVO4)	Leakage of 221Fr (1st daughter of 225Ac) and 213Bi (3rd daughter of 225Ac) was 55.4 ± 3.6%, and 25.6 ± 1.5%, respectively (5 days) Leakage of 221Fr and 213Bi was of 41.1 ± 2.5% and 19.8 ± 1.16%, respectively (11 days) Leakage of 223Ra 73.0 ± 4.0% (4 days), and 30.0 ± 1.7% (33 days) The leakage of 211Pb was 44.8 ± 3.1% (35 days)	---	---	[24]
	Gadolinium vanadate core (Gd(225Ac)VO4) and core + 2 shell (Gd(225Ac)VO4/2GdVO4)	Leakage of 225Ac was 15% and 2.4% from core and core + 2 shells Leakage of 227Th was 3% and 1.5%, from core and core + 2 shells, respectively Leakage of 221Fr (first decay daughter) from core was 40.6 ± 2.4% (2 days) and ~69.5 ± 4.9% (23 days) Leakage for 213Bi from core and core + 2 shell was 22.5 ± 1.3% and 19.6 ± 1.9%, respectively Leakage of 227Th was ~3% (15 days) and 1.6 ± 0.3% (18 days) Leakage of 223Ra from core was 10.0 ± 0.7% (1 day) and 39.0 ± 2.2% (2 weeks)	Yield of 225Ac was 77.1 ± 13.2% and 96.6 ± 1.6% for core and core + 2 shell, respectively Yield of 227Th was 62.9 ± 2.7% and 81.9 ± 1.2% for core and core + 2 shell, respectively	3.6 ± 0.9 nm for core and 4.4 ± 1.0 nm for core + 2 shell	[79]
	Gd0.8Eu0.2VO4 core and core + 2 shells	The leakage of 221Fr from core was 40.9 ± 0.3% (2 days), 55 ± 3.6% (10 days) and 67.6 ± 3.3% (28 days) Leakage of 221Fr from core + 2 shells was 36.3 ± 6.2% (10 days) and 45.5 ± 3.6% (28 days) The leakage of 213Bi was <15% for core and ~22% for core + 2 shells	Yield core was 41.1 ± 16.5%, shells increased the yield up to 55%	6.1 ± 1.4 nm for core and 12.4 ± 2.0 nm for core + 2 shells	[80]
	Liposomes: Pegylated liposomes membrane charge (zwitterionic and cationic)	213Bi retention was 10% (650 nm) 225Ac retention in the zwitterionic liposome was ~88% (30 days) 225Ac retention in cationic liposomes was 54%	6.4–10.0%	200/400/650 nm	[11]
	Liposomes	225Ac retention of 81 ± 7%	55–73%	121 ± 6 nm	[81]
	Liposomes	Retention of 225Ac with up to three 225Ac nuclides per every 2 liposomes	58.0–85.6%	107 ± 2 nm	[82]
	Multivesicular liposomes (MUVELs)	Retained 98% of 225Ac (30 days) Retention of 213Bi was 31% (758 nm)	---	---	[83]
	Polymersomes	Retention 221Fr was 71.5% (800 nm, 24 h) Retention 213Bi was 74% (800 nm, 24 h)	213Bi was 83 ± 75% (100 nm) 225Ac was 67 ± 0.8% (100 nm)	100, 200, 400, and 800 nm	[28]
	Double-layered polymersomes	Retention of ~100% of 221Fr (800 nm) Retention of 213Bi (recoil atoms) was 80% (800 nm)	---	300–800 nm	[26]
	Polymersomes	Retention of 225Ac was 92 ± 3% Retention of 221Fr (first daughter) was ~20% Retention 213Bi was ~10%	89 ± 0.6%	100/200/400/800 nm	[84]
	Polymerosomes	---	>90% for 111In and >64% for 225Ac	---	[85]
	Polymerosomes	221Fr and 213Bi were retained at the tumor site 88 ± 9% and 89 ± 2%, respectively (2 days p.i.)	54–59%	97 ± 37 nm	[86]
	Fullerenes: 225Ac metallofullerene	---	---	---	[87]
	TiO2	Retained >95% of 225Ac and 221Fr (10 days)	99.8 ± 2.1%	25 nm	[88]
	Carbon nanotubes	Clear rapidly and shown to be therapeutically effective in vivo	---	---	[89]
	Carbon nanotubes	---	~95%	---	[90]
	Lipid vehicle	225Ac were retained to significant extents by during 6 h	62.7 ± 14.6%	106 ± 4 nm	[91]
223Ra	LnPO4 core and core + 2 shells NPs (Ln = La, Gd)	The core LaPO4 NPs retained up to 88% of 223Ra (35 days) Retention of 99.9% of 223Ra and 211Pb (daughter of 223Ra) the core + 2 shells (27 days) Retention ~100% of 211Pb (decay daughter) Retention of 99.98% of 225Ra/225Ac parents and ~80% for the 221Fr and 213Bi daughters in the core + 2 shells (35 days)	91% for LaPO4 core + 2 shell	3.4 nm for core and 6.3 nm for core + 2 shells	[75]
	Hydroxyapatite (HA)	No significant release of activity was detected	>95%	15 nm	[92]
	Hydroxyapatite (nHAp) and titanium dioxide (nTiO2)	The sorption efficiency on nHAp was 95 ± 5% and approximately 100% in the case of nTiO2	~95%	---	[93]
	Hydroxyapatite (HAP)	---	98%	Width up to 100 nm and length up to 500 nm	[94]
	Hydroxyapatite (HAP)	---	98%	900–1000 μm	[95]
	Liposomes	223Ra retention was 96 ± 1% (24 h) and 93 ± 2% (100 h) 228Ac retention was 95 ± 2% (24 h)	223Ra was 78 ± 6% 228Ac was 61 ± 8%	---	[96]
	Liposomes: Pegylated liposomal doxorubicin (PLD)	Liposomal 223Ra was relatively stable in vivo and was largely retained in liposomes	51–67%	80 nm	[97]
	223/224/225Ra-nanozeolite (NaA)	The leakage in PBS solutions of 224,225Ra-nanozeolites was below 0.5%	99.9%	30–70 nm	[22]
	223Ra-labeled nanozeolite	The leakage of 223Ra was <0.5% Release of 211Pb and 211Bi (decay products) was 2% (1 day) and 5% (6 days) in human serum Retention of 90–95% of 211Pb and 211Bi (decay products)	99.9%	50–80 nm	[76]
	BaSO4	---	20%	140 nm	[98]
	Superparamagnetic iron oxide nanoparticles Fe3O4 SPIONS	PBS, bovine plasma, and serum showed a cumulative release of 223Ra below 5%	85–99% (PBS)	4–26 nm	[99]
	Reduced graphite oxide	Desorption in PBS + BSA solution <5% of 99mTc Desorption in BSA in PBS <10% of 99mTc Desorption in PBS + BSA solution in 1 h raised to 35% for 90Y	Sorption was 10% for 223Ra, 90% of 99mTc, 80–100% for 207Bi and 90Y	4–6 nm	[100]
	Polyoxopalladates (Pd-POM): [224Ra]Na-a(Ra)Pd15	224Ra was incorporated into [224Ra] Na-Ba(Ra)Pd15, but the insertion is not selective (lead and bismuth Pd-POM can also be generated)	---	---	[101]
	Calcium carbonate microparticles	Retention >95% for 224Ra and its daughter nuclide 212Pb (1 week)	>80% of 224Ra and 212Pb (daughter nuclide)	---	[102]
211At	Silver core coated by PEO shell	---	50–97%	18.3–34.7 nm	[73]
	Ultrashort nanotubes	Retention was 19.6% and 11.0% for water and methanol, respectively	77.7–91.3%	20–50 nm in length and 1 nm diameter	[103]
	Gold nanoparticles (AuNPs)	---	>99%	5 and 15 nm	[104]
	Gold nanoparticles (AuNPs)	---	>99%	5 nm	[105]
212Pb	Liposomes	---	75%	---	[106]
	Liposomes	212Pb and 212Bi were 95% retained (20 h)	90 ± 2%	---	[107]
	Hydroxyapatite (HAP)	---	---	---	[108]

The {Gd_0.75 La_0.25}(²²⁵Ac)PO₄@4 LaPO₄ shell@Au nanosystem showed 100% of ²²⁵Ac retention over three weeks. The ²²⁵Ac retention did not do not show variation compared with {La_0.5Gd_0.5}PO₄@GdPO₄@Au doped with ²²⁵Ac, but ²²¹Fr retention varied from 60–89% as a function of time, the number of layers, and nanoparticle composition [78]. Later, in 2014, presented the second-generation layered nanoparticles {La_0.5Gd_0.5}(²²⁵Ac)PO₄@4 GdPO₄ shell@Au, the ²²⁵Ac retention was almost 100%; however, the ²²¹Fr retention was only 60% in in vitro studies. This second generation achieved more than 70% of ²¹³Bi retention in the lungs and approximately 90% at 1 and 24 h after injection [77]. The authors concluded the ability to retain the ²²⁵Ac and its decay daughters in the NP core depends on its composition and the type and number of added shells [31,79,80].

Edyta et al. (2018) proposed titanium dioxide (TiO₂) nanoparticles (25 nm diameter) as ²²⁵Ac carrier and their decay products. NPs retained more than 95% of ²²⁵Ac and ²²¹Fr over 10 days in PBS and physiological salt and with a yield of 99.8 ± 2.1% [90]. In 2020, Suchánková et al. studied ²²³Ra sorption on hydroxyapatite (nHAp) and titanium dioxide nanoparticles (nTiO₂). The sorption efficiency on nHAp was 95 ± 5% and approximately 100% in the case of nTiO₂ [95].

In 2014, Kozempel et al. evaluated the in vitro stability and labeling yield hydroxyapatite nanoparticles (HA-NPs) radiolabeled with ²²³Ra. In their studies, no significant release of activity was detected on stability tests, probably due to resorption of released ²²³Ra and ²¹¹Pb or due to the decay of the daughter nuclides of short-lived. The labeling yield of ²²³Ra was greater than 95% for intrinsically labeled but did not provide data on the release or retention of daughter nuclides [94]. Later, in 2016, Vasiliev et al. synthesized hydroxyapatite nanoparticles with a width of up to 100 nm and a length of 500 nm, as carriers of ²²³Ra with a yield of up to 98% [96]. Kukleva et al., in 2006, studied the uptake of ²²³Ra in the superparamagnetic nanoparticles (SPION) of Fe₃O₄ (diameters between 4 and 26 nm). The in vitro results showed yields greater than 85% in PBS, and the stability tests in PBS, bovine plasma, and serum showed a cumulative release of ²²³Ra below 5% [101]. Westrøm et al., in 2018, proposed calcium carbonate microparticles as carriers of ²²⁴Ra. The results show that both ²²⁴Ra and its daughter nuclide ²¹²Pb were adsorbed with yields greater than 80% and reported retention of more than 95% for both radionuclides for up to 1 week in vitro. Biodistribution studies showed that the highest activity of ²¹²Pb (²²⁴Ra daughter with the longest half-life) was found in the kidneys, mainly [104]. Reissig et al. (2019) proposed functionalized BaSO₄ nanoparticles as carriers to ²²⁴Ra. The NPs had sizes of approximately 140 nm, and it was possible to incorporate 20% of ²²⁴Ra into the NPs [100]. Gott et al. proposed the use of polyoxopalladates (Pd-POM), in which Ra²⁺ can be easily introduced into the nucleus. The results showed that ²²⁴Ra was incorporated into [²²⁴Ra] Na-Ba(Ra)Pd_15, but the insertion is not selective, as lead and bismuth Pd-POM can also be generated [103]. Toro-González et al., in 2020, synthesized gadolinium vanadate (GdVO₄) NPs sizing <100 nm as platforms for ²²⁵Ac, ²²³Ra, and ²²⁷Th encapsulation. They reported the leakage of ²²¹Fr (1st daughter of ²²⁵Ac) and ²¹³Bi (3rd daughter of ²²⁵Ac) as 55.4 ± 3.6% and 25.6 ± 1.5% after 5 days in dialysis, and after 11 days it decreased to 41.1 ± 2.5% and 19.8 ± 1.16%, respectively. In the case of ²²³Ra and ²¹¹Pb (decay daughters of ²²⁷Th), they showed that the leakage of ²²³Ra (1^st decay daughter) reached a maximum of 73.0 ± 4.0% after 4 days, and then decreased to 30.0 ± 1.7% at 33 days finally in ²¹¹Pb (3rd decay daughter of ²²³Ra). The leakage of ²¹¹Pb showed that the leak increased from 11.1 ± 1.0% to 44.8 ± 3.1% after 35 days on dialysis [24].

There have been extensive theoretical and experimental investigations on the possibility of using liposomes for testing the retention of recoils in the decay chains of ²²⁵Ac [11,83,84,85,98] and of ²²³Ra [98,99]. Sofou et al. (2004) prepared pegylated liposomes of different membrane charges (zwitterionic and cationic) and sizes to entrap ²²⁵Ac with mean encapsulation efficiency between 6.4% and 10.0%. This efficiency translated into 10–40 actinium atoms per liposome. In this research, the theoretical and in vitro fraction of ²¹³Bi retention as a function of liposome size and composition was evaluated. Theoretical calculations have revealed that for satisfactory ²¹³Bi retention (>50%), liposomes of relatively large sizes (>650 nm in diameter) are required. Experimentally, several studies have verified ²¹³Bi retention to be liposome size dependent. The ²¹³Bi retention was significantly higher in the 800 versus the 100 nm zwitterionic liposomes. However, not even the mother radionuclide ²²⁵Ac was completely retained, and ²¹³Bi retention was 10% for the largest liposomes (650 nm). Regarding the composition of the liposomes, ²²⁵Ac retention in the zwitterionic liposome fractions was more than 88% over 30 days. While in cationic liposomes, ²²⁵Ac retention was above 54%. The authors proposed that the liposomal encapsulation of ²²⁵Ac to retain the daughters is size dependent [11]. Multivesicular liposomes (MUVELs) were prepared by Sofou et al. in 2007 to increase daughter retention using the MUVELs, which are large liposomes with entrapped smaller lipid vesicles (rigid membranes composed of diheneicosanoyl phosphocholine and cholesterol) containing ²²⁵Ac. PEGylated MUVELs retained 98% of encapsulated ²²⁵Ac for 30 days. Retention of ²¹³Bi (the last daughter) was 31% in the MUVELs, with an average size of 758 ± 287 nm. As in the previous work, the authors did not observe higher daughter retention attributable to the larger size of the carrier (1 µm diameter). Due to the limitations of stable intact liposomes having such large diameters, it is important to evaluate new materials to develop in vivo stable outer large shell encapsulations [85]. Henriksen et al. (2004) prepared liposomes containing ²²³Radium and ²²⁵Ac, and the stability and the retention of these radionuclides in serum were evaluated. The loading of ²²³Radium within liposomes was 78 ± 6% with retention of 96 ± 1% after 24 h and 93 ± 2% after 100 h. The loading of ²²⁸Ac (²²⁵Ac was not available at the time of the experiments) was 61 ± 8%, and the retention was 95 ± 2% after 24 h [98]. Later, in 2008, Chang et al. (2008) proposed various conditions for loading high activities of ²²⁵Ac using pegylated liposomes of 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (DNPC) and cholesterol after showing in a previous study that liposomes can stably retain encapsulated ²²⁵Ac for long periods. Liposomes were encapsulated with three ²²⁵Ac nuclides per every two liposomes with a diameter of 121 ± 6 nm, a loading efficiency of 55–73%, and ²²⁵Ac retention of 81 ± 7% of the initially encapsulated radioactivity was achieved. The main release of ²²⁵Ac from all liposomes can retain and remain encapsulated for 30 days. The study showed that ²²⁵Ac is loaded to a higher yield in preformed liposomes and is more stably retained over time when the ionophore liposomes were loading with citrate buffer [83]. Bandekar et al. reported anti-PSMA liposomes with 107 ± 2 nm of diameter loaded with ²²⁵Ac to selectively kill prostate-specific membrane antigen (PSMA) expressing cells. The loading efficiency of ²²⁵Ac into liposomes ranged from 58.0% to 85.6%. The targeted and un-targeted PSMA liposomes exhibited similar retention of encapsulated ²²⁵Ac with up to three ²²⁵Ac nuclides per every two liposomes. However, their study did not report the retention of ²²⁵Ac or any of its daughter radionuclides [84]. Jonasdottir et al. in 2006 reported the first phase II study of alpha-emitting radioliposomes with a loading efficiency of between 51% and 67% of ²²³Ra in pegylated liposomal doxorubicin (PLD), which had an average diameter of approximately 80 nm. The comparison between liposomal ²²³Ra versus free cationic ²²³Ra showed that the liposomal ²²³Ra was catabolized, and the ²²³Ra released was either excreted or absorbed by the skeleton. This skeletal uptake increased up to 14 days after treatment, but ²²³Ra in bone was lower in the group given liposomal ²²³Ra versus cationic ²²³Ra, and spleen and bone received the highest absorbed doses. The authors concluded that liposomal ²²³Ra was relatively stable in vivo and that ²²³Ra was largely retained in liposomes [99]. Henriksen et al. (2003) incorporated ²¹²Pb into preformed liposomes and evaluated the in vitro retention of it and its radioactive daughter ²¹²Bi during incubation in serum, the yield of ²¹²Pb within the liposomes was 90 ± 2% after 30 min. ²¹²Pb and ²¹²Bi were 95% retained in the liposomes after 20 h of incubation [108].

Kruijff et al. succeeded in synthesizing [²²⁵Ac]InPO₄ nanoparticles by coprecipitation within the polymersomes, with a loading efficiency of 89 ± 0.6% and retention of ²²⁵Ac of around 92 ± 3%, while retention of ²²¹Fr (first daughter) was approximately 20%, and ²¹³Bi retention of approximately 10% was reported [86]. Wang et al. evaluated the percentage of recoil daughters that are retained in polymersomes of different sizes upon encapsulation of ²²⁵Ac and ²¹³Bi. Results showed a loading efficiency of 83 ± 5% and 67 ± 0.8% for ²¹³Bi and ²²⁵Ac, respectively, in polymersomes of 100 nm. The highest experimental retention values for ²²¹Fr (71.5%) and ²¹³Bi (74%) were achieved in the largest polymersomes (800 nm), 24 h after loading. These results indicate that larger polymersomes are needed to attain satisfactory retention of recoiling radionuclides [28]. Thijssen et al. (2012) carried out a study based on computer simulations (Monte Carlo simulations code “NANVES”) describing the best polymersome design for the retention of the recoil daughters of ²²⁵Ac (²²¹Fr and ²¹³Bi). Simulations showed that the best polymersome design comprises a double-layered vesicle with the nuclide encapsulated inside the innermost layer and that the size is also important due to larger polymersomes retaining recoils better. For instance, double-layered vesicles with a diameter of 800 nm are capable of retaining ²²¹Fr completely and approximately 80% of the ²¹³Bi recoil atoms, while the escape of ²¹³Bi is reduced to 20% and the percentage of alpha particles emitted from escaped daughter products outside the nanocarrier is less than 10%. The double-layered polymersomes with outer diameters of 300 and 400 nm retained 41% and 47% of the ²¹³Bi recoils, respectively [26]. Kruijff et al. (2018) demonstrated polymersomes radiolabeling efficiencies > 90% for ¹¹¹In and > 64% for ²²⁵Ac. The distribution of the daughter atoms in vivo was not assessed [87].

There also investigations where carbon nanomaterials have been proposed for ²²⁵Ac, ²²³Ra, and ²¹¹At radionuclides [89,91,92,102,105]. Gold nanoparticles conjugated to ²¹¹At [105,106], liposomes with ²¹²Pb/²¹²Bi [107,108], and hydroxyapatite nanoparticles [109] for retention of ²¹²Pb have been proposed with limited success.

9. Discussion

Targeted alpha therapy to this day remains a very promising therapeutic option for cancer due to the localized cell destruction that originates from high LET and short ranges of alpha particles. However, achieving complete retention of the decay daughters of some alpha emitters remains the biggest challenge to be solved. The main strategies in targeted therapies to limit the nuclear recoil effects are focused on the use of nanocarriers that can stop the propagation of recoil and provide a controlled release. In this way, the effect of radiation exposure to healthy tissues by free daughter radionuclides formed can be significantly reduced. The nanosystems presented in this review provide an account of the potential of the retention and behavior of radionuclides within nanocarriers, and their performance depends on the different materials used. In general, small nanoparticles usually have better biodistribution and faster clearance than bigger ones [23], but in some cases, in systems such as liposomes and polymersomes, where the retention of decay daughters was measured, they demonstrated size retention effects [11,28,83,84,85,86,87,98,99]. Although the larger sizes perform better in terms of retention of decay daughter, the larger sizes pose severe problems as sizes beyond 150 nm are ineffective in penetrating tumor vasculature and are also less efficient extravasation. Therefore, a trade-off in liposomal size and retention power is important in enhancing retention effects and enhanced patency in the uptake of tumors. In the case of inorganic nanoparticles, it was shown that small nanoparticles can safely confine the decay daughters that occur in a cascade of alpha particles. Strategies suggested by Woodward et al. (2011) [76] and McLaughlin et al. [31,79,80], using small core nanoparticles that are loaded with alpha emitters and then surrounded by confining shells preferentially with high-Z materials, appear to be the most promising.

However, it is still challenging to make accurate predictions on the size and thickness of surrounding shells.

Although some systems show better retentions in vitro than others, the characteristics that the nanocarrier system must meet to completely contain the children of the disintegration chains have not been found to satisfactory outcomes or predictability. Therefore, more detailed investigations are imminent to gain insights on the most optimum nanomaterials capable of providing optimum retention of alpha emitters for ultimate utility in oncology.

10. Conclusions

In this review, we have summarized the latest advances in targeted alpha-particle therapy (TAT) through relevant and pertinent literature citations. We have discussed promising results in terms of various systems showing optimum in vitro retention, with limited success so far in achieving optimum in vivo characteristics of TAT. We have provided a strong and scientifically justifiable rationale for the application of nanotechnology in transforming targeted alpha-particle therapy optimally effective in cancer therapy. In this context, we have discussed the importance and the interrelationships of the chemical nature, size, and types of nanomaterials (singular carbon-based, metallic vs. liposomal) on the overall in vivo efficacy of targeted alpha-particle therapy. The data to date suggest that the retention and confinement of alpha-emitting radionuclides do not always depend on the size of nanoparticulate systems. It is important that the nanoparticulate-embedded alpha emitters exhibit sizes within the 50–150 nm range to be able to penetrate the tumor vasculature more effectively. Inorganic core nanoparticles loaded with alpha emitters and surrounded by confinement layers seem to be the most efficient alternative, although more research is needed to find the appropriate thickness of the confinement layers, thus opening up future new research endeavors in TAT.