Targeted Radium Alpha Therapy in the Era of Nanomedicine: In Vivo Results

Targeted alpha-particle therapy using radionuclides with alpha emission is a rapidly developing area in modern cancer treatment. To selectively deliver alpha-emitting isotopes to tumors, targeting vectors, including monoclonal antibodies, peptides, small molecule inhibitors, or other biomolecules, are attached to them, which ensures specific binding to tumor-related antigens and cell surface receptors. Although earlier studies have already demonstrated the anti-tumor potential of alpha-emitting radium (Ra) isotopes—Radium-223 and Radium-224 (223/224Ra)—in the treatment of skeletal metastases, their inability to complex with target-specific moieties hindered application beyond bone targeting. To exploit the therapeutic gains of Ra across a wider spectrum of cancers, nanoparticles have recently been embraced as carriers to ensure the linkage of 223/224Ra to target-affine vectors. Exemplified by prior findings, Ra was successfully bound to several nano/microparticles, including lanthanum phosphate, nanozeolites, barium sulfate, hydroxyapatite, calcium carbonate, gypsum, celestine, or liposomes. Despite the lengthened tumor retention and the related improvement in the radiotherapeutic effect of 223/224Ra coupled to nanoparticles, the in vivo assessment of the radiolabeled nanoprobes is a prerequisite prior to clinical usage. For this purpose, experimental xenotransplant models of different cancers provide a well-suited scenario. Herein, we summarize the latest achievements with 223/224Ra-doped nanoparticles and related advances in targeted alpha radiotherapy.


Introduction 1.Targeted Alpha Radiotherapy
Because the shortcomings of conventional radiotherapy [1][2][3] limit the attractiveness of standard oncological treatment approaches, attempts have been made to optimize the delivery of radiation therapy to cancer cells.Recently, targeted radionuclide therapy (TRT) using alpha emitters has gained remarkable attention for cancer treatment [4].
Due to their favorable physicochemical properties, alpha emitter radionuclides are capable of exerting cellular cytotoxicity on tumor cells, thus causing minimal exposure to the healthy tissues nearby [5].Complex DNA disruptions [6,7] induced by the high linear energy (LET: 50-230 keV/µm) of alpha rays are responsible for a significant tumor cell-killing effect, while the short path of the particles (50-100 µm) ensures the protection of the surrounding intact organs [5,8].Beyond DNA damage, alpha particles cause direct alterations to other cellular compartments, such as the mitochondria, lysosomes, and the cell membranes, which, along with bystander and immunological effects, have a central role in the achievement of alpha-mediated anti-tumor effect [9][10][11].The short penetration within tissue favors the eradication of micrometastatic and residual lesions as well as single malignant cells [12].Attached to target-specific carriers (for example, antibodies, peptides, or small molecules), alpha emitters are able to selectively irradiate all neoplastic lesions, including the primary tumors as well as their metastases irrespective of their location [13][14][15].In addition, the radiotoxicity of high-LET alpha particles is independent of oxygenation; therefore, radioisotopes with alpha emission are considered suitable for destroying tumor masses even under hypoxic conditions [16,17].Overall, these characteristics validate that alpha particles have greater biological effectiveness compared to other radiations in therapeutic settings.
Experiments with preclinical model systems represent the cornerstone to gaining deeper insight into the mechanism of action of TRT and the assessment of long-term treatment response.Findings derived from such investigations are not only transferable to in-human patient care but may also lay the groundwork for the identification of novel cellular targets for radiation purposes.The therapeutic value/suitability of targeted alpha treatment was evidenced by a vast array of prior in vitro and in vivo studies [5,[18][19][20][21][22]. Within the alpha-emitting radionuclide domain, a broad set of isotopes are available for TRT usage, including, for instance, Radium-223 ( 223 Ra), Radium-224 ( 224 Ra), Actinium-225 ( 225 Ac), Thorium-227 ( 227Th ), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Lead-212 (212Pb), and Astatine-211 ( 211 At) [16].Figure 1 provides statistics from the current clinical trials and the number of preclinical and clinical publications on 223 Ra, 224 Ra, 225 Ac, 227Th , 212 Bi, 213 Bi, 212 Pb, and 211 At isotopes.
within tissue favors the eradication of micrometastatic and residual lesions as well as single malignant cells [12].Attached to target-specific carriers (for example, antibodies, peptides, or small molecules), alpha emitters are able to selectively irradiate all neoplastic lesions, including the primary tumors as well as their metastases irrespective of their location [13][14][15].In addition, the radiotoxicity of high-LET alpha particles is independent of oxygenation; therefore, radioisotopes with alpha emission are considered suitable for destroying tumor masses even under hypoxic conditions [16,17].Overall, these characteristics validate that alpha particles have greater biological effectiveness compared to other radiations in therapeutic settings.
Experiments with preclinical model systems represent the cornerstone to gaining deeper insight into the mechanism of action of TRT and the assessment of long-term treatment response.Findings derived from such investigations are not only transferable to inhuman patient care but may also lay the groundwork for the identification of novel cellular targets for radiation purposes.The therapeutic value/suitability of targeted alpha treatment was evidenced by a vast array of prior in vitro and in vivo studies [5,[18][19][20][21][22]. Within the alpha-emitting radionuclide domain, a broad set of isotopes are available for TRT usage, including, for instance, Radium-223 ( 223 Ra), Radium-224 ( 224 Ra), Actinium-225 ( 225 Ac), Thorium-227 ( 227Th ), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Lead-212 (212Pb), and Astatine-211 ( 211 At) [16].Figure 1 provides statistics from the current clinical trials and the number of preclinical and clinical publications on 223 Ra, 224 Ra, 225 Ac, 227Th , 212 Bi, 213 Bi, 212 Pb, and 211 At isotopes.A,B) and the number of preclinical and clinical publications (C,D) on 223 Ra, 224 Ra, 225 Ac, 227Th , 212 Bi, 213 Bi, 212 Pb, and 211 At isotopes.(A) The percentage distribution (%) of different alpha-emitting radionuclides in clinical studies-available to date-investigating the anti-tumor efficacy of alpha radiation: 223 Ra (84%), 224 Ra (0%), 225 Ac (6%), 227Th (0%), 212 Bi (0%), 213 Bi (1%), 212 Pb (2%), and 211 At (7%).Data obtained from clinicaltrials.gov.(B) Detailed status analysis of the clinical trials using the investigated alpha-emitting radionuclides.Data obtained from clinicaltrials.gov.(C) Distribution of the published preclinical and clinical scientific papers in the field of alpha-emitting radionuclide therapies in the last 10 years.Data obtained from pubmed.ncbi.nlm.nih.gov.(D) The number of preclinical and clinical publications on targeted alpha radionuclide therapies from the last 5 and 10 years.Data obtained from pubmed.ncbi.nlm.nih.gov. 223.Ra: Radium-223, 224 Ra: Radium-224, 225 Ac: Actinium-225, 227Th : Thorium-227, 212 Bi: Bismuth-212, 213 Bi: Bismuth-213, 212 Pb: Lead-212, 211 At: Astatine-211.A,B) and the number of preclinical and clinical publications (C,D) on 223 Ra, 224 Ra, 225 Ac, 227Th , 212 Bi, 213 Bi, 212 Pb, and 211 At isotopes.(A) The percentage distribution (%) of different alpha-emitting radionuclides in clinical studies-available to date-investigating the anti-tumor efficacy of alpha radiation: 223 Ra (84%), 224 Ra (0%), 225 Ac (6%), 227Th (0%), 212 Bi (0%), 213 Bi (1%), 212 Pb (2%), and 211 At (7%).Data obtained from clinicaltrials.gov.(B) Detailed status analysis of the clinical trials using the investigated alpha-emitting radionuclides.Data obtained from clinicaltrials.gov.(C) Distribution of the published preclinical and clinical scientific papers in the field of alpha-emitting radionuclide therapies in the last 10 years.Data obtained from pubmed.ncbi.nlm.nih.gov.(D) The number of preclinical and clinical publications on targeted alpha radionuclide therapies from the last 5 and 10 years.Data obtained from pubmed.ncbi.nlm.nih.gov. 223.Ra: Radium-223, 224 Ra: Radium-224, 225 Ac: Actinium-225, 227Th : Thorium-227, 212 Bi: Bismuth-212, 213 Bi: Bismuth-213, 212 Pb: Lead-212, 211 At: Astatine-211.With regard to the positive physicochemical features of alpha-emitting Radium (Ra) isotopes, tremendous research has been undertaken to explore their capability in targeted therapeutic settings [23][24][25].Due to its high skeletal affinity, Ra is widely used in the treatment of bone metastases [26,27]. 223Ra, for example, has been successfully applied for alleviating bone pain due to metastases in breast or prostate cancer patients [28].Figure 2A,B demonstrate the decay characteristics of two Ra isotopes: 223 Ra and 224 Ra.With regard to the positive physicochemical features of alpha-emitting Radium (Ra) isotopes, tremendous research has been undertaken to explore their capability in targeted therapeutic settings [23][24][25].Due to its high skeletal affinity, Ra is widely used in the treatment of bone metastases [26,27]. 223Ra, for example, has been successfully applied for alleviating bone pain due to metastases in breast or prostate cancer patients [28].Figure 2A,B demonstrate the decay characteristics of two Ra isotopes: 223 Ra and 224 Ra.Out of the radionuclides of Ra, 224 Ra, seems to be a potential candidate for therapeutic purposes as an alpha emitter [23,[29][30][31].Previous clinical research revealed pain reduction Out of the radionuclides of Ra, 224 Ra, seems to be a potential candidate for therapeutic purposes as an alpha emitter [23,[29][30][31].Previous clinical research revealed pain reduction induced by 224 Ra treatment in patients with ankylosing spondylitis [32,33].So far, however, there is little research related to the application of 224 Ra in oncological settings [30,34].It could be acquired from Thorium-228 ( 228 Th) generators [35].Compared to other short-lived alpha radioisotopes, such as 213 Bi (T 1/2 = 46 min), 211 At (T 1/2 = 7.2 h), and 212 Pb (T 1/2 = 10.6 h), the longer half-life of 224 Ra (3.631 (2) days) enables easy transport to distant medical facilities; hence, the establishment of an on-site radiochemical laboratory is not a prerequisite for 224 Ra production [36,37].Being an alpha emitter, 224 Ra possesses high LET values and a short tissue range, which are necessary for the induction of an adequate radiotoxic effect. 224Ra is often called a nanogenerator, as it decays to Lead-208 ( 208 Pb) via six daughter nuclides involving Radon-220 ( 220 Rn), Polonium-216 ( 216 Po), 212 Pb, 212 Bi, Polonium-212 ( 212 Po), and Thallium-208 ( 208 Tl) [38].Decaying through the emission of four alpha and two beta particles with total alpha energies of approximately 26 MeV [31,39], 224 Ra allows for the transport of sufficient doses to the target areas with low amounts of administered radioactivity.Moreover, the alpha-emitting decay progenies of 224 Ra (Thoron/220-Radon/ 220 Rn; 216 Po; 212 Pb/ 212 Bi; 212 Po) could also have meaningful therapeutic implications, which might contribute to the enhancement of the overall antitumor effect [40,41].In addition, the limited accessibility of 211 At, and the high amount of therapeutically required relevant doses of 213 Bi, also speak in favor of the application of 224 Ra [42,43].The decay scheme of 224 Ra is displayed in Figure 2B.
Beyond 224 Ra, 223 Ra is also emerging as a valuable radionuclide for targeted tumor treatment [44,45].Acting like a calcium mimetic, 223 Ra shows increased complexation with skeletal calcium hydroxyapatite crystals in areas of active bone formation [46,47]. 223Ra could be eluted from Actinium-227/Thorium-227 ( 227 Ac/ 227 Th) generator system or it can be obtained from uranium mill tailings [48].Its multiple-step decay (4 α, 2 β and 5 γ rays) leads to the following emitted energy distributions: 26.87 MeV, 0.94 MeV, and 1.996 MeV for α, β and γ particles, respectively [49]. Figure 2A presents the decay series of 223 Ra.The high LET value of 223 Ra ensures that it exerts its cytotoxicity through the induction of doublestrand DNA breaks [50].Similarly to 224 Ra, due to the high LET value (approximately 28 MeV) and the short path of 223 Ra alpha rays (60-100 µm), relatively small radioactive doses are sufficient for the achievement of substantial therapeutic effect [24].This is of paramount importance for both personnel and patients regarding the reduction in their exposure to radiation.Similarly to daughter 224 Ra, the alpha rays of 223 Ra decay products could also be therapeutically exploited ( 219 Rn, 215 Po, 211 Bi), thereby leading to improved anti-tumor-killing effect.Its broad accessibility compared to other alpha emitters such as 225 Ac, 211 At, or 213 Bi, along with the economically viable synthesis from 227 Ac generators, allow the performance of a wide array of preclinical experiments as well as clinical trials possible [51,52].The long half-life (T 1/2 = 11.43 days) of 223 Ra and the relatively large amounts obtained upon a single elution ensure easy shipment to distant locations, which further contributes to the commercial distribution and the centralized production of both the isotope and its radiopharmaceuticals [53][54][55].In a practical sense, the longevity of 223 Ra also gives enough room for synthesis, radiolabeling, handling, and quality control [53,56]; moreover, it could be seamlessly customized to the half-time of larger molecules including peptides or nanoconjugates.
More recently, in an attempt to overcome the delivery challenge, nanoparticles have become embraced as carriers for radioisotopes.In addition to oncological applications, however, Radium-carrying nanoprobes could also be exploited as antiviral agents [65].Based on the pioneering results of Pijeira et al., for example, folic acid-functionalized graphene quantum dots (GQD-FA) seem to be useful tools against Zika virus (ZIKV) infection; moreover, the 223 Ra-labeled radioactive counterpart ( 223 Ra@GQD-FA) may also broaden the therapeutic avenue of tumors with folate-receptor upregulation [65].
Figure 2 shows the decay chains of 223 Ra (Panel A) and 224 Ra (Panel B).

Nanoparticles for Targeted Radionuclide Delivery
Nanoparticles with an intrinsic ability to transport cancer therapeutics are an emerging category of nanoscale platforms that holds great promise in targeted tumor treatment [66][67][68].A wide spectrum of nanoparticles is currently available; examples include but are not limited to polymeric nanoparticles, liposomes, micelles, quantum dots, carbon nanotubes, dendrimers, magnetic nanoparticles, and solid lipid nanoparticles [69].The application of anti-tumor agents packaged into nanocarriers could bypass the limitations of traditional oncological therapies, including unspecific targeting, accidental death of healthy cells, and the development of systemic side effects or drug resistance [70,71].In addition to chemotherapeutic drugs, small molecule inhibitors, peptides, proteins, nucleic acids (RNAi) and radioisotopes can be loaded into nanoparticles.
Nanomaterial-based cytotoxic medications are reported to be superior to either free drugs or other therapeutic molecular conjugates in several facets [70,72].First, the attachment of the therapeutic agents to different nanoparticles guarantees more selective transport to target sites coupled with enhanced solubility, bioavailability and tissue permeation that ultimately leads to improved overall anti-tumor therapeutic gain [67,[72][73][74].By choosing the size of the nanoparticle, the tumor retention and therefore the therapeutic effect of the delivered cargo could be flexibly regulated, which constitutes another advantage of the use of nanoparticles as carriers [72,75].More specific targeting along with lengthened tumor residence time ensure improved killing effect at the disease sites.The size and the shape of the nanoparticles along with the chemical properties of the surface coating are the key determinants of tumor uptake and related therapeutic effect [76][77][78][79].For this reason, these factors must be taken into account during the de novo construction of nanomaterials to attain a maximal anti-tumor effect with negligible systemic toxicity.Based on the latest literature data, the addition of biocompatible materials onto the surface of the nanomaterials influences their biodistribution and tumor accumulation and therefore the therapeutic efficacy [79,80].Affecting the interplay between nanoparticles and serum proteins/cell membranes, surfaces charges have a major role in the determination of the organ uptake pattern and the circulatory half-time of the nanomaterials [79,81,82].In a prior study of Arvizo and co-workers-for example-nanoparticles with neutral (hydroxyl group-terminated, −1.1 mV) and zwitterionic (quaternary ammonium and sulfate groupterminated, −2.0 mV) surface charges appeared to have prolonged blood half-time coupled with higher uptake in the tumor sites [80].In addition to these, the internalization of polyethylene glycol (PEG) and dextran into the surface of the nanoagents serves as another way to develop highly biocompatible molecules [83,84].
Nevertheless, the facile adjustment of drug extravasation from the nanoconjugates to its mechanism of action also serves the achievement of the required anti-tumor effect [72].Compared to their free matches, nanocarrier-drug formulations provide more aggravated drug absorption into the tumor, which further contributes to attaining desirable cytotoxicity [70].In addition-compared to other drug transporters such as antibodies-they have the potential to deliver larger amounts of molecules [85,86] promoting elevated drug concentration levels at the lesions of interest.Importantly, neither the type nor the number of the transported particles modify their organ distribution or pharmacokinetics.Moreover, nanoparticles not only make the delivered drugs resistant against degradation [70], but owing to their endocytosis-mediated cellular entrance, they are not prone to multidrug resistance (MDR) processes, either [72].In comparison with free therapeutic counterparts, nanocarrier-engulfed drugs are protected even against any interplay with the biological microenvironment [70], which together with the above-listed benefits has a major role in the maximization of the therapeutic effect.By the same token, the nanocarrier coverage promotes drug stability enhancement as well as the extension of the circulatory half-time, thereby aiding the establishment of a favorable safety profile [87,88].
Since the size of the nanoparticles makes the encapsulation of several ligands with distinct receptorial target sites possible, nanomaterials also allow for the development of multivalent delivery systems [89].Characterized by higher receptor binding affinity, multireceptor targeted nanosystems can cover an extended subset of cell surface receptors, which apart from the improvement of therapeutic efficacy contributes to better diagnostics during treatment follow-up.In addition, the self-assembly of nanostructures enables the construction of multifunctional nanosystems with improved biocompatibility and biodegradability that could be exploited in targeted anti-cancer or even in gene drug delivery [90,91].Furthermore, self-assembled nanostructures including nanoparticles, nanotubes, nanofibers, or hydrogels are characterized by adjustable bioactivity, variable surface chemistry, and target specificity that along with the low production costs also make them emerge not only as promising drug carriers but also as useful tools for different biological purposes such as tissue regeneration or bioimaging [92,93].Since the assembly of separate nanoparticles results in the merge of their inherent properties, the regulation of self-assembly leads to the development of complex nanoplatforms with a wide range of characteristics [94][95][96].In addition to the regulation of conventional nanoparticle assembly, the control of nanoparticle accumulation and disintegration-denoted as reversible self-assembly-may continue to broaden the horizon of nanomaterial applications in medical fields [94].Furthermore, bispecific nanosystems make simultaneous target-specific treatment and imaging possible.For example, the use of a magnetic bispecific cell engager (MagBICE)-composed of biodegradable iron nanoparticles and antibodies that join the therapeutic cell to the treated cell (Bispecific antibodies)-combines therapy and magnetic imaging [97,98].Integrating photodynamic therapy and immunotherapy, NJ nanoparticles (prodrug of NLG919 and JQ1) coated with photosensitizer pyropheophorbide-a (PPa)-modified PHP (PHPNJ) are also promising examples for bispecific nanoparticles [99].
Overall, considering the advanced therapeutic indices and the simultaneous reduction in treatment-related side effects [72] along with the above-detailed encouraging results, the translation of nanosystems into further stages of research seems straightforward.
Despite these beneficial properties, there are some potential downsides and challenges that pose an issue on their routine application.Given the possible toxicity of nanocarriers, the assessment of in vitro and in vivo cytotoxic and immunotoxic activity is a prerequisite for usage [56,100].Although the evaluation of their in vivo behavior is an area ripe for further research, available data suggest that the physicochemical characteristics of nanomaterials-for example size or surface area-that affect their binding properties to blood components, elimination kinetics and the cross-talk with cellular and endothelial barriers largely determine their in vivo fate.Consequently, it impacts their ability to enter different regions/body compartments, which could be associated with potential toxic effects [72,101].There are a couple of papers reporting the safety of different NaA nanozeolite nanomolecules including NaA-silane-PEG or its prostate-specific membrane antigen (PSMA) targeting match-NaA-silane-PEG-D2B-in normal prostate cell lines (RWPE-1, HPrEC) and in prostate cancer cell lines (LNCaP C4-2, DU-145) [56,102].Comparably, no obvious signs of in vitro toxicity were observed in association with several nanoparticles such as nanozeolites A, nanozeolites Y, pure silica nanozeolite silicalite-1, unaltered NaA nanozeolite, and Barium-133-labeled nanozeolite A (BaA) attached to poly(ethylene glycol) (PEG) of long chain (BaA-silane-PEGm(MW1000), BaA-silane-PEGm(MW2000)) [103][104][105].Based on literature data, toxicogenomics serves as a promising tool to explore nanocarrierassociated immunotoxicity and related processes such as hypersensitivity, immunogenicity, autoimmunity, and immunosuppression [56,106,107].In contrast to the above-mentioned information, Lankoff et al. [56] could not draw a conclusion regarding the in vitro immunotoxic effects of NaA nanozeolite nanocarriers (NaA-silane-PEG and NaA-silane-PEG-D2B) because they experienced the deregulation of both the pro-and anti-inflammatory genes induced by these nanocarriers [56,108,109].For this reason, relevant studies are required to uncover those aspects of nanoparticle-related toxicity that are poorly understood yet.
Albeit meaningful progress has been made toward the understanding of how nanoformulations interreact with the environment, the complete discovery of the way these compounds pass through the tumor mass still remains part of future research [72,110].In this respect, it is therefore worthwhile to continue exploring the role of physiological barriers, tumor heterogeneity and hypoxia, endo-lysosomal escape mechanism, as well as the way of elimination [67] to fully elucidate and improve their mode of action.In addition, to impede too early dissociation of the drug from the nanoconjugates, the optimization of release kinetics should be another important step that would definitely increase the translational potential of nanosystem-based therapeutics to in-human application.Further shortcomings may arise from the costs and technical hurdles of the synthesis of nanomaterials.
The use of carriers guarantees prolonged residence time for the Ra isotopes in the target location and therefore allow for enhanced therapeutic effect at the disease sites.Thus, they not only serve the purpose of ensuring Ra uptake beyond the skeletal system, but they also have a remarkable contribution to the improvement of anti-tumor radiotherapeutic effect.In addition, microparticles may also aid in reducing the untoward radiation of non-target organs by retaining much of the release of Ra decay products derived from the nuclear recoil effect [60,122].
In this review, we discuss the therapeutic potential of 223/224 Ra-doped nanoparticles in preclinical models to present an up-to-date overview of recent advances in targeted alpha radiotherapy.

Overview of In Vivo Results with 223/224 Ra-Labeled Nanoparticles
Considering the above-mentioned characteristic of Ra radionuclides, the construction of 223 Ra/ 224 Ra-based nanoparticles has attracted the attention of several researchers.Ample amount of preclinical experiments were directed toward the radiosynthesis, the radiochemical analysis and the in vitro characterization of 223 Ra-labeled nanocarriers [123][124][125][126]. Based on these results, several nanoparticles bound to 223 Ra seem to be suitable for further in vivo biomedical applications including hydroxyapatite, titanium dioxide (TiO 2 ), Fe 3 O 4 superparamagnetic iron oxide nanoparticles (SPIONs), barium ferrite nanoparticles (BaFeNPs), gadolinium vanadate nanoparticles (GdVO 4 ), barium sulfate (BaSO 4 ), detonation nanodiamonds (NDs), reduced graphite oxide (rGiO), multi-walled nanotubes (MWCNTs), and radium dichloride nanomicelles ([ 223 Ra]-RaCl 2 ) [61,[123][124][125][126][127][128][129][130].Experiencing satisfying in vitro stability for [ 223 Ra]Fe 3 O 4 nanoparticles in various biological niche such as PBS, bovine plasma, and serum, Mokhodoeva et al. strengthened their potential for in vivo MRI, nuclear imaging and theranostic purposes [124].The results of Suchánková et al.according to which both the intrinsic labeling and the surface labeling of hydroxyapatite and titanium dioxide nanoparticles with 223 Ra resulted in labeling yields greater than 94%confirm the use of these nanomaterials as effective radioisotope transporters in in vivo applications [125].In addition, using HER2 receptor-positive SKOV-3 cells [127], 223 Ralabeled BaFeNPs attached to trastuzumab (Herceptin ® , [ 223 Ra]BaFe-CEPA-trastuzumab) exhibited excellent targeting capability, cell internalization and cytotoxicity that projects BaFeNPs as promising radionuclide carriers in nuclear medicine.Moreover, results on the sorption of various radionuclides ( 9m Tc, 207 Bi/ 213 Bi, 90 Y, 226 Ra/ 223 Ra) on different carbon nanomaterials (CNMs: NDs, rGiO and MWCNTs) as well as findings on their controlled release from the CNM surface area indicate the feasibility of CNMs for radiopharmaceutical development [128].Although further steps need to be taken to enhance radionuclide retention inside the GdVO 4 NPs, given the partial encapsulation of 223 Ra (~75%) in GdVO 4 NPs and its maximum leakage of 73.0 ± 4.0%, GdVO 4 NPs are also viable alternatives for isotope transport [129].Finally, the higher cytotoxic effect of [ 223  Although Ra-labeled nanoparticles are still in their early stages of application in in vivo preclinical imaging, preliminary results indicate great promise [43,56,102].Table 1 presents an overview of the preclinical studies on Ra-labeled nanoparticles.Investigating the uptake pattern of Radium-224 ( 224 Ra), Westrøm et al. performed biodistribution studies with the free cationic form of 224 Ra ( 224 RaCl 2 ) and with 224 Ra-labeled calcium carbonate microparticle (CaCO 3 ) suspensions (0.4 mL) of three different particle types in healthy nude mice 1, 4, and 7 days post-intraperitoneal (ip.) tracer administration (as seen in Table 1) [112].First-generation, second-generation and PlasmaChem microparticles with diameters from 3 to 15 µm, 1 to 3 µm, and 1 to 3 µm, respectively, were prepared by the spontaneous precipitation method [131].All ip.organs exhibited radioactivity after the injection of the first-generation 224 Ra-CaCO 3 including the liver, the stomach, the intestines, the parietal peritoneum, and the ip.fat tissue, except for the spleen, which showed signs of tracer concentration post- 224 RaCl 2 application (both labeled in Dulbecco's PBS with 0.5% BSA).Although due to the affinity of 224 Ra to the bones, the highest free 224 Ra accretion was present in the skeletal system (skull, femur), the osseous uptake of the first-generation 224 Ra-CaCO 3 significantly decreased, which indicated moderate release of the isotope from the microparticle (both labeled in Dulbecco's PBS with 0.5% BSA).Interestingly, 224 Ra efflux from the smallest PlasmaChem (labeled in sucrose) microparticles was reported to be more enhanced.The skeletal uptake of 224 Ra-which is proportional to the release of the isotope from the microparticle-shows negative association with the radioactivity that remained in the peritoneum, which is of central importance in terms of the ip.therapeutic effect of the isotope.Since varying ip. 224Ra retention could impact the radiotherapeutic efficacy, appropriate chelation is essential to achieve targeted cytotoxicity.
Of note, faint cardiac, cerebral and muscle activity was displayed for both the free 224 Ra and the first-generation radiopharmaceuticals (both labeled in Dulbecco's PBS with 0.5% BSA) [112].The higher lienal radioactivity for the first generation agent-labeled in sucrose solution-compared to that of the counterpart labeled in Dulbecco's PBS with 0.5% BSA or that of the PlasmaChem could be accountable to the activity of the peritoneal fat measured in the close vicinity of the spleen.

Tumor Growth Inhibition in Preclinical Models of ES-2 Tumors
In addition to SKOV-3-luc tumors, a couple of prior studies reported on the therapeutic efficacy of 224 Ra-CaCO 3 in the targeted treatment of ES-2 ovarian tumors [31,43].Given the lengthened median survival and decreased amount of ascites of the irradiated ES-2-transplanted mice compared to the saline-injected controls, Westrøm and co-workers proved the therapeutic power of 224 Ra-CaCO 3 in an ovarian tumor model of more advanced disease stage as well [43].Upon the ip.administration of four different types of 224 Ra-CaCO 3 , the same research group led by Napoli managed to achieve similar survival benefit in ES-2 tumor-bearing mice to that of Westrøm and co-workers; moreover, they concluded that the experienced therapeutic success was not dependent on either the labeling method or the addition of polyacrylic acid (PAA) coating to the microparticles [132].The research of Napoli et al. (2021) [132] is summarized in Table 1.
Similarly to the SKOV3-luc models, Westrøm et al. did not find any significant difference between the survival length of the ES-2 xenotransplanted mice of the different treatment cohorts either (150, 300, 1000, or 2 × 150 kBq/kg); therefore, we may conclude that neither dose increment nor repeated treatments influence the anti-tumor potential of 224 Ra-CaCO 3 particles.The advanced stage of the neoplasms, inferior peritoneal dispersion of the 224 Ra-CaCO 3 microparticles and their rapid clearance from the abdominal cavity could underpin the lack of dose dependency [43].Additionally, the loss of p53 protein which has a central contribution to radiation regulation may also be responsible for the lack of the development of dose-dependent radiosensitivity [133][134][135][136]. Transcription factor p53 has a central role in mediating cellular response to radiation [134].DNA damage induced by radiation exposure triggers the accumulation p53 protein [137,138], which activates cellular pathways related to apoptosis, cell death or DNA repair mechanisms and cell survival [139,140].Therefore, the loss of p53 or alterations in its mechanism of action may be associated with cancer development and resistance to chemo-and radiotherapy [141][142][143].Although recent data confirmed the inevitable contribution of p53 to radiation control, its role is largely dependent upon the type of the irradiated organs and cells [140].In addition to the above-mentioned information, however, microparticle aggregates may preclude alpha particles from being evenly distributed in the peritoneum, which could constrain the effective irradiation of some target tissues and organs [31].Furthermore, the short-lived nature of 224 Ra and related shorter tumor retention time may also provide some possible explanations.Correspondingly to the results of Westrøm et al., Larsen and colleagues did not find a relationship between the dose of the administered radioactivity and the therapeutic response either, applying 211 At-labeled monodisperse polymer particles ( 211 At-MDPP) in a preclinical ip.K13 hybridoma model [144].In their study, identical survival was observed upon the administration of 7, 65, 200 or 500 kBq of 211 At-MDPP to Female Balb/c mice ip.xenotransplanted with K13 hybridoma cells [144].
Similarly to Westrøm et al., Li and colleagues also assessed the anti-cancer capability of 224 Ra-CaCO 3 in ES-2 ovarian tumor models of female athymic nude-Foxn1 nu mice (presented in Table 1) [31].In light of the improved survival, Li et al. further validated the therapeutic success of the isotope-loaded microparticle (0.4 to 4.6 kBq/mg of 224 Ra-CaCO 3 ), which was in corroboration with the findings of Westrøm et al. and Napoli et al. [31,43,132].Albeit notably lengthened survival was detected in the group administered with the free cationic form of 224 Ra ( 224 RaCl 2 ) compared to the untreated animals, the best therapeutic outcome achieved by the carrier-coated probe ( 224 Ra-CaCO 3 ) indicates the therapy-enhancing effect of the CaCO 3 microparticle (survival rates in the control, 224 RaCl 2 , and 224 Ra-CaCO 3 -treated cohort: 0/9, 1/5, and 10/10, respectively) [31].In a like manner, earlier findings of Larsen et al. with free 211 At and 211 At-MDPP also demonstrated the significance of microparticles in exploiting the radiotherapeutic effect of the transported isotopes [144].Radionuclides bound to nano/microparticles have extended the residence time in the tumor niche compared to the free matches that is essential for the exertion of sufficient cytotoxic effect. 224Ra-CaCO 3 , which requires 25% lower radioactivity, has the same anti-tumor efficacy as 224 RaCl 2 , which indicates less radiation exposure associated with 224 Ra-CaCO 3 handling.All this is of utmost importance regarding its integration into standard patient care [31].In a similar way, this favorable safety profile was authenticated by Westrøm et al. [43].We assume that the therapeutic gains of 224 Ra-CaCO 3 coupled with the reduced radiation danger will drive its optimization and the continuing investigation into further stages of clinical research prior to being an approved product for cancer patients.
The therapeutic indices measured by Li and colleagues in the low (240-300 kBq/kg), intermediate (380-460 kBq/kg), and high-dose (940-1140 kBq/kg) treatment groups with corresponding values being 1.5, 1.8 and 2.8 suggest a kind of dose-response relationship between the administered radioactivity and the therapeutic outcome [31].This may correlate with the findings of Westrøm and co-workers, who noted a similar trend; however, no statistically significant direct association was confirmed between the injected dose and the success of the therapy [43].Varying treatment setups or tumor stages could be supposed as possible explanations for this difference between the two studies.Unlike Westrøm et al., who noted reduced femoral 224 Ra-CaCO 3 activity (meaning reduced systemic release) with increasing mass doses that led to improved therapeutic outcome, Li et al. did not point out a similar negative correlation [31,112].As the skeletal uptake is positively associated with isotope release form the nanoparticle and therefore is inversely proportional to the radiotherapeutic effect at the target site, factors that affect radionuclide efflux-other than mass doses-must be further investigated.Furthermore, the remarkable reduction in ascites volume registered by Li et al. in relation to the treatment active group compared to the controls (mean volume: 1.4 vs. 4.7 in the treated and in the untreated cohorts, respectively) is closely associated with the observations of Westrøm and co-workers [31].
As the diffusion of Thoron/220-Radon ( 220 Rn)-one of the decay progenies of 224 Rafrom the pores [131] of the microparticles, is reported to enhance anti-tumor ability, Napoli et al. aimed to investigate the effect of the way of radiolabeling on 220 Rn release with ES-2 xenograft models [132].Applying surface absorption or inclusion-labeled CaCO 3 microparticles, Napoli et al. proved that the different labeling procedures neither influence the release of 220 Rn from the microparticles nor the anti-tumor effect of the probes in the ES-2 tumor-carrying mice [132].Inclusion-labeled 224 Ra-CaCO 3 microparticles with a PAA coating, however, were associated with moderately lower anti-tumor potential, which was presumably due to the presence of the polymer layer that led to the decrease in 220 Rn release from the microparticles [132].Table 1 gives a brief summary of the most important points of their study.
Based on the findings obtained, it can be established that more consistent irradiation of the peritoneum induced by the efflux of 220 Rn may aid in overcoming the limitations deriving from inappropriate 224 Ra-microparticle distribution [132] and subsequent dose inhomogeneity, which could result in remarkable therapeutic enhancement.Furthermore, 220 Rn emanation can generate the expansion of the penetration depth of alpha particles, making their transport beyond the general alpha particle traveling distance possible.It means that Radon-220 diffusion extends the penetration range of alpha-emitting Radium-224, and this contributes to the therapeutic efficacy of 224 Ra-labeled nanoparticles.Owing to the 3-fold higher mean diffusion length of 220 Rn (300-400 µm) compared to the maximum pathway of an alpha particle (100 µm) in water, an extended volume could be irradiated via the release of the first progeny of 224 Ra- 220 Rn [132].By the same token, diffusing alphaemitters radiation therapy (DaRT) that is based on the emission of the alpha-emitting decay products of 224 Ra is proposed as a novel method of targeted cancer treatment [29,[145][146][147].With the application of 224 Ra-labeled wires embedded into experimental squamous cell carcinoma tumors, Arazi et al. demonstrated a necrotic area with a diameter of 5-7 mm corresponding to the irradiated region, which supported the anti-tumor efficacy of progenyrelated radiation [29].Accordingly, initial trials also affirmed the therapeutic advantages of the radiation stemming from daughter nuclei in DaRT under clinical circumstances in the treatment of locally advanced and recurrent squamous cell carcinoma (SCC) of the skin and the head and neck [147,148].
To make sure that alpha rays reach the lesions of interest, apart from 220 Rn efflux, the microparticles must also be localized close to the tumor to be irradiated.This is also of pivotal importance for the maintenance of constant irradiation.

Toxicity Evaluation
The absence of 224 Ra-CaCO 3 -induced histopathological cytotoxicity coupled with no signs of irreversible, treatment-related hematological and clinical chemical abnormalities project the safety of the compound, which is of utmost importance regarding its transportation to clinical usage [43].The toxicity profile of 224 Ra-CaCO 3 is summarized in Table 2. Correspondingly to the observations of Westrøm et al., neither did Li et al. encounter any acute or subacute toxic effects in association with the 224 Ra-CaCO 3 treatment of ES-2 xenotransplanted mice (as shown in Table 2) [31].On the contrary, another research study of Westrøm and co-workers revealed that 224 Ra bone uptake is inversely proportional to the injected dose, which indicated the possibility of bone marrow toxicity [112].In addition to this, toxic effects coming from the redistribution of the decay products of 224 Ra should also be addressed [132,[149][150][151]. Through reporting hepatic and red blood cell 212 Pb (T 1/2 = 10.64 h) overaccumulation along with renal 212 Bi (T 1/2 = 60.54 min) uptake in beagles administered with 224 Ra, Lloyd et al. drew the attention to the potential organ toxicity of 224 Ra [149,152].In a similar way, progeny-related adverse effects were published by Milenic and co-workers who investigated the acute and long-term effects of ip. or intravenously (iv.) injected 212 Pb-TCMC-trastuzumab [150].The relevant findings of their study are displayed in Table 2. Seven or 90 days post-injection of Balb/c mice with 212 Pb-TCMC-trastuzumab (ip.and iv.: 0.0925-1.85MBq and 0.0925-1.11MBq, respectively), 212 Pb-associated toxicity was observed in the bone marrow, spleen, kidneys, and liver, which was confirmed by histopathological examinations (demonstrated by Table 2) [150].According to Wick and Gössner's observation, the radiation of healthy tissues-induced by the release of daughter nuclei-could lead to renal dysfunction and tumor development such as skeletal malignancies or chronic myeloid leukemia.These observations of theirs perfectly correlated with prior findings [153].By registering more elevated renal 212 Pb accretion for the first generation 224 Ra-CaCO 3 particles than for the free 224 Ra (dissolved RaCl 2 ), Westrøm et al. also corroborated the redispersion of the isotope from the target site to remote organs and tissues [112].These results highlight the importance of the integration of tissue/organ toxicity assessment in the design of future studies with radiolabeled nanoparticles.In addition to the appearance of unwanted toxicity-given the meaningful contribution of the alpha-decaying nuclides of 212 Pb to the alpha energy release of 224 Ra-its reallocation to organs far from the point source may reduce the overall therapeutic dose [132,145,154,155].Therefore, to mitigate treatment-related adverse effects and to maintain ideal therapeutic outcome, attempts should be made to diminish the efflux of progenies.As part of liquid phase experiments, Napoli et al. noted the adsorption of 212 Pb by 224 Ra-CaCO 3 microparticles, which in part may possibly reduce the redistribution of the daughter radioisotope [132].In line with this, based on the in vitro retention studies of Westrøm et al., the mean retained 212 Pb radioactivity was reported to exceed 95% in case of PlasmaChem (5 µm) and first-generation 224 Ra-CaCO 3 (18 µm) microparticles that were radiolabeled in sucrose solution and maintained in the same medium [112].We may conclude that the particle size as well as the way of particle synthesis influence the radioisotope retention of the nanoparticles.Despite all this, previous in vivo research comparing α-emitter-based ip.treatment with β therapy proved that the former ones performed better regarding anti-tumor and/or toxicity aspects [20,156,157].Although performed with antibodies as carriers, pioneering clinical trials even demonstrated negligible toxicity in relation to ip.-administered α-radiotherapy [158][159][160].
In contrast to previous preclinical and clinical findings, the lack of treatment-related particle residuum [43] authenticated the biodegradable and biocompatible nature of CaCO 3 [161,162].Two weeks post-intracavital administration of 90 kDa poly(lactic-coglycolic) acid (PLGA) micro, and nanoparticles (5-250 µm) to male SV129 mice, Kohane and colleagues noted a meaningful presence of permanent residue, adhesions, histopathological signs of chronic inflammation and foreign body giant cells [161].Identically, within the framework of prior organ uptake studies on 224 Ra-CaCO 3 , microparticle remnants were depicted by Westrøm et al. in immunodeficient healthy female athymic nude Foxn nu mice after the ip.injection of different types of radiolabeled CaCO 3 microparticles [112].Based on necropsy experiments, they found a positive correlation between the quantity of the peritoneal leftovers and the amount of the injected microparticles.Remnants smaller than a diameter of 3 mm were related to CaCO 3 microparticles between 1 and 5 mg, whereas postinjection of microparticles of 25 mg, larger residuum were encountered.The observations of an earlier in-human phase I study with 12 gynecological cancer patients reported polymer residues adhesions, fat necrosis, and foreign body giant cell development in association with the administration of paclitaxel microspheres, which also contradicted the results of Westrøm et al. [43,162].
In addition, we must consider the fact that-like radioactive radiation in generalthe positive and negative effects of the therapeutic isotopes depend on the taxonomic classification of the irradiated animal (mouse, rat, dog) and, within that, on the strain and species.It is also known from the literature that within the strains, the radiation effect (toxic, therapeutic) induced by either external or internal radiation sources is greatly influenced by the gender, age, immunological status, repair mechanisms (e.g., DNA repair) as well as the status of the cell cycle and oxygenation of the irradiated tissues, etc. [163-165].

Clinically Transportable Findings
Westrøm et al. depicted a positive correlation between the SKOV3-luc tumor bulks and the neutrophil granulocyte-to-lymphocyte ratio (NLR), which may be related to worse survival rates [43,166].Considering the relationship between elevated NLR in ovarian cancer and increased level of tumor marker CA-125, sizeable amount of ascites, more severe disease stage and poorer treatment response, the follow-up of NLR throughout chemotherapy/radiotherapy should raise attention [166].Presumably due to progressed disease conditions, notably higher white blood cell (WBC) levels were measured in the control and in the 150 kBq/kg 224 Ra-CaCO 3 -receiving ES-2 bearing mice, indicating the value of WBC as a prognostic biomarker besides NLR.Therapy-related reduction in ascites volume experienced in mice carrying ES-2 tumors [43] is supposed to be an outstanding therapeutic benefit, since based on prior literature findings, the volume of ascites is regarded as an independent determinant of survival in ovarian cancer [167].Therefore, we suppose that regular blood count monitoring, in particular leukocytes and their subtypes, as well as the measurement of ascites volume should be part of the management of patients under targeted radiotherapeutic treatment.These parameters would not only provide a representation of the current disease state but also would help to assess the response to therapy that could be used to select those patients who would benefit from further treatment.

223 Ra-Labeled Nanomaterials
Aside from 224 Ra, the formerly detailed favorable characteristics of 223 Ra render increasing interest for its application in the design of target-specific nanocarriers.As previously mentioned, significant efforts have been made to find the best-fitting nanomaterial or chelator to attach 223 Ra to target-specific molecules [115,116,120,121,125,126]. 223 Ra-labeled Prostate-Specific Membrane Antigen (PSMA)-Targeting Nanoparticle ( 223 RaA-silane-PEG-D2B) According to the in vitro results of Czerwi ńska and Lankoff, 223 Ra-labeled prostatespecific membrane antigen (PSMA)-targeting NaA nanozeolites decorated with anti-PSMA monoclonal antibody D2B ( 223 RaA-silane-PEG-D2B) could be potential future candidates for the targeted radio-treatment of PSMA receptor-positive prostate cancer (PCa) patients [56,102].Table 1 presents a resumé on the study of Czerwi ńska and Lankoff.In vitro binding studies with 131 I-PSMA-PEG-silane-NaA, internalization experiments and MTTassay-based metabolic activity measurements-performed in PSMA-expressing LNCaP C4-2 cells (human lymph node prostate carcinoma cell line) [168] and in PSMA-negative DU-145 cells (human epithelial prostate carcinoma cell line) [169]-demonstrated selective receptor binding, rapid and specific cellular internalization and substantial cytotoxicity in PSMA overexpressing LNCaP C4-2 cells for 223 RaA-silane-PEG-D2B, and this projected its therapeutic feasibility under in vivo circumstances as well (displayed in Table 1) [102].

In Vitro Results with
The results of the annexin/propidium iodide assay also verified the capability of the PSMA-affine nanocarriers in the induction of cell death by showing an increase in the number of the early apoptotic and late apoptotic/necrotic human primary prostate epithelial cells (HPrEC).In a like manner, the more increased number of caspase 3/7 positive apoptotic HPrEC cells related to the administration of NaA-silane-PEG and NaA-silane-PEG-D2B compared to the control cells receiving PBS further strengthened apoptosis induction caused by these compounds (NaA-silane-PEG: 2.5% vs. 1.4% and NaA-silane-PEG-D2B: 3.7% vs. 1.4% for apoptotic and control cells; respectively) [56]. 223RaA-silane-PEG and 223 RaA-silane-PEG-D2B Despite the promising in vitro apoptosis induction capability, the considerable activity of 223 RaA-silane-PEG and 223 RaA-silane-PEG-D2B in off-target organs such as the liver, lungs, spleen, and the bones of PSMA-positive LNCaP C4-2 tumor bearing mice may hamper the application of these derivatives in imaging settings.Moreover, their negligible tumor accumulation coupled with the meaningful background noise limits the precise delineation of the neoplastic mass from the neighboring healthy organs.Hence, biochemical modifications are required to optimize the pharmacokinetics of the nanoconjugates for diagnostic purposes as well.Even though D2B exerts outstanding PSMA selectivity in vivo [170], the low Enhanced Permeability and Retention effect (EPR) of the antibody-impregnated radioconjugate ( 223 RaA-silane-PEG-D2B) may impede optimal tumor accumulation [171].Similarly, association between the EPR effect and the tumor uptake of different nanoparticles was reported in prior preclinical studies with breast, ovarian, pancreatic cancer, and squamous cell carcinoma xenografts [172][173][174].Thus, special emphasis should be laid on the enhancement of EPR to prolong tumor retention time and delay nanoparticle leakage from the tumor mass [171,172].In addition, although 223 RaA-silane-PEG-D2B with a diameter of 250 nm [82] is fitted to the pore size of the subcutaneously developing LNCaP C4-2 prostate cancer, it shows continuous change during tumor progression that impacts EPR effect and related tumor accumulation [175][176][177].To enhance EPR and thus the therapeutic efficacy of nanoparticles, the size and shape of the nanoagents along with the chemical properties and charge of the surface coating must be addressed and selected carefully [76][77][78][79]178]. Since the blood perfusion makes a critical contribution to the transport of nanoparticles to tumor sites through the EPR [179], the use of drugs such as angiotensin II for blood flow enhancement in the target area could be a possible solution for the improvement of EPR.Furthermore, adjuvants, inflammatory factors, or antibody photosensitizers seem to be feasible to increase drug transport via altering the EPR effect [180][181][182][183].

In Vivo Biodistribution of
The exact mechanism behind the experienced high non-target uptake [56] is not yet fully elucidated; nevertheless, the activity of the hepatic, pulmonary and lienal reticuloendothelial (RES) cells could be supposed in the respective organs.According to prior pharmacokinetic findings, the hydrodynamic size, the water solubility, and the presence of "bio-corona" coverage on the surface of the nanoparticles are the major determinants of the cellular uptake and the organ distribution pattern of nanomaterials [173,[184][185][186]. Owing to the adherence of the serum components to the outer layer of the administered nanomolecules, a protein-lipid coat-termed as "bio-corona"-is formed on the surface of the nanoconjugates, which is associated with the nanoparticle size [173,186].Given the more pronounced phagocytotic activity of RES cells in case of nanomaterials of larger hydrodynamic size, the rapid uptake of 223 RaA-silane-PEG (approx.196 nm) and 223 RaAsilane-PEG-D2B (approx.250 nm) by the macrophages leads to increased accumulation in the corresponding organs [102,173].The results of Lankoff et al. could be consistent with those of previous studies that confirmed high concentrations of the subsequent nanocarriers in organs related to the RES system: 99m Tc-Fe 3 4 -1-PNPs-hEGFR (99m Technetium ( 99m Tc)-labeled poly(ethylene glycol) (PEG)-based polymeric nanocarriers (PNPs) with iron oxide (Fe 3 O 4 ) nanoparticles and human epithelial growth factor receptor (hEGFR) and 177 Lu@Fe 3 O 4 -CEPA or 177 Lu@Fe 3 O 4 -CEPA-trastuzumab nanoconjugates ( 177 Lu-labeled supermagnetic iron oxide nanoparticles attached to human epidermal growth factor receptor type 2 (HER2) antibody trastuzumab) [173,187].
According to existing literature data, the in vivo experienced higher skeletal accumulation of 223 RaA-silane-PEG and 223 RaA-silane-PEG-D2B [56] could be primarily attributed to the release of 223 Ra and its daughter nuclides from the microparticles and their reassimilation to the bones [60,112,188].As opposed to this, the in vitro observations of the same research group showed insignificant isotope efflux from the investigated nanomolecules in human serum [102] that may be caused by the differences between the in vitro and in vivo experimental circumstances.Since in vitro studies are performed under controlled conditions, they may fail to correctly represent the physiological mechanisms/functions/processes of a living organisms.On the other hand, in vivo investigations are accomplished in whole living organisms that resemble better the characteristics of organs and tissues; hence, they supply more reliable results on functioning.Therefore, the results of in vitro studies may not correspond to those of the in vivo ones.In addition, the other free isotope of radium in the form of 223 RaCl 2 exhibited lower skeletal uptake compared to the detailed nanoconjugates with greater particle size ( 223 Ra-silane-PEG-D2B and 223 Ra-silane-PEG), which could possibly be attributed to the shorter residence time of 223 Ra in the bones due its smaller size as well as the reallocation of the radionuclide from the mentioned nanoparticles (Alpharadin (Xofigo) Assessment Report; 2021). 223Ra-Labeled Prostate Cancer-Targeting Nanoparticles In line with the experience of Li et al., Lankoff and co-workers did not find evidence for therapy-triggered body weight loss either one day after the administration of the unlabeled nanocarriers (NaA-silane-PEG and NaA-silane-PEG-D2B) and the radioconjugates ( 223 RaAsilane-PEG and 223 RaA-silane-PEG-D2B) into healthy BALB/c mice.This indicated the lack of treatment-related acute or subacute toxicity (presented in Table 1) [56].Although similarly to the findings of Westrøm et al. and Li et al., no signs of treatment-associated toxicity were noted 24 h post-injection of either 223 RaA-silane-PEG, 223 RaA-silane-PEG-D2B or their unlabeled derivatives during the follow-up of body weight, various laboratory parameters and histopathological specimens, a week after treatment with the 223 Ra-labeled compounds, bone marrow fibrosis and a meaningful decrease in the platelet counts and body weight reduction were found in the irradiated study mice [31,43].The hematotoxicity of 223 RaAsilane-PEG and 223 RaA-silane-PEG-D2B was further validated upon the comparison of the one-day and seven-day blood count results, which revealed a significantly deteriorating blood count profile regarding WBC, red blood cell and platelet counts, hematocrit and hemoglobin concentrations as the therapy progressed [56,102].Consistent with the findings of Lankoff et al., in the phase II clinical trial of Hofman and colleagues, grade 3-4 thrombocytopenia was observed in 27% of castration-resistant prostate cancer (CRPC) patients administered with another PSMA-targeting radioprobe (Lutetium-177 ( 177 Lu)-PSMA) [189].In addition, several clinical studies also revealed comparable hematological toxicity related to targeted 177 Lu-PSMA-617 treatment in prostate cancer sufferers [190][191][192][193].In contrast, Westrøm et al. showed no treatment-induced hematological toxicity even 44-45 days after ip. 223Ra-CaCO 3 treatment in SKOV3 tumor-bearing mice (day 0: inoculation; day 3: treatment; day 47-48: blood sampling); however, in the ES-2 xenotransplanted cohort, a discrete platelet number reduction was encountered post- 223 Ra-CaCO 3 administration that was not statistically meaningful.Based on these observations, we may conclude that treatment with radium-labeled nanoparticles may affect hematopoiesis; hence, the monitoring of blood counts is recommended during such therapies.Emphasis must be placed on the fact; however, that while Czerwi ńska and Lankoff performed toxicity assessment in a group of healthy mice, Westrøm et al. used tumor-bearing animals for the experiments, which may raise some difficulties regarding the direct comparison of the results of the two studies.In addition, biochemical analyses showed elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) for all nanoconjugates (NaA-silane-PEG, NaA-silane-PEG-D2B, 223 RaA-silane-PEG and 223 RaA-silane-PEG-D2B) regardless of the presence of radiolabeling [56], which did not match the results of Westrøm et al. and therefore was probably not 223 Ra-irradiation-related but rather due to the hepatic accretion of the compounds.Identically, in the study of Sun et al., Sprague-Dawley rats intratracheally administered with silica nanoparticles (SiNPs) also exhibited more increased ALT and AST serum concentrations compared to the untreated animal group [194].

Alpha Targeting of Prostate Cancer beyond 223-Radium ( 223 Ra)
Currently, apart from 223 RaCl 2 , there is no other molecule in clinical usage for the targeted therapy of mCRPC [195].In addition to 223 Ra, the anti-tumor potential of other alpha emitters including 225 Ac has also been explored in the treatment of mCRPC patients [196].Based on the drop of prostate-specific membrane antigen (PSA) levels, and complete therapeutic response on [ 68 Ga]Ga-PSMA-11 PET/CT scans, Kratochwil et al. reported the efficacy of 225 Ac-labeled PSMA small molecule inhibitor PSMA-617 ([ 225 Ac]Ac-PSMA-617) in patients with an aggressive type of PCa [193].Identically, Sathekge et al. experienced an outstanding reduction in PSA concentrations (from 237 to 43 µg/L) after the application of two series of [ 213 Bi]Bi-PSMA-617; however, unlike [ 225 Ac]Ac-PSMA-617, no treatmentinduced adverse health ramifications were perceptible [197].
Although further modifications are required for pharmacokinetical optimization, the results of Czerwi ńska and Lankoff provided a strong and scientifically justifiable rationale for the future integration of 223 RaA-silane-PEG and 223 RaA-silane-PEG-D2B into the treatment armamentarium of PCa.

223-Radium ( 223 Ra)-Labeled Liposomes
Given the in vivo stability and the favorable organ uptake pattern of 223 Ra-labeled liposomes, these nanomaterials could also serve as useful tools for the targeted transport of 223 Ra in therapeutic settings [121].Upon the biodistribution assessment of 223 Ra-coated pegylated liposomal doxorubicin ( 223 Ra-PLD) in healthy Balb/C mice, Jonasdottir et al. found the highest tracer concentration in the liver and in the spleen, which-similarly to 223 RaAsilane-PEG and 223 RaA-silane-PEG-D2B [56]-could have been caused by the phagocytic activity of the RES cells.Table 1 provides a brief overview of the study of Jonasdottir et al.According to initial ex vivo findings, the splenic radiotracer accretion in laboratory mice was higher compared to that of the dogs [121].The slow blood clearance of 223 Ra-PLD was evidenced by meaningful blood activities from the early time points (1 and 24 h) as well as the presence of relatively low tracer concentration in the kidneys [121].In line with this, extremely high liposomal 223 Ra localization indices (LIs) were registered for the blood, which also supported the prolonged and even the much slower circulatory clearance of the nanoparticle compared to free 223 Ra.The blood half-life of the liposome-attached 223 Ra was reported to be more than 24 h.It was in correlation with that of Caelyxì/Doxilì liposome formulation in rodents (24-35 h) and in dogs (23-27 h) [198], whereas the half-life of dissolved 223 RaCl 2 in the blood was significantly lower: approximately an hour.Contrary to this, Westrøm et al. did not encounter any differences between the blood elimination of free 224 Ra and that of the 224 Ra nanoconjugate ( 224 Ra-CoCO 3 ) [112].In agreement with the previous results of Westrøm et al. and Lankoff et al. [56,112], the redistribution of 223 Ra to the bones from the radioactive liposome ( 223 Ra-PLD) could be responsible for the experienced increased femoral and cranial radiotracer uptake [121].Although recent studies demonstrated that bones are resistant to 223 Ra-induced bone marrow toxicity to an unexpectedly high extent [47,199], given the long circulatory half-life of the radioactive liposomes along with the large number of nanoparticles in the blood, the potential occurrence of skeletal cytotoxicity must be expected.This finding indicates the importance of the evaluation of bone marrow toxicity and the tracking of related hematological laboratory parameters during in vivo preclinical treatment with liposomal 223 Ra or even in human application.In addition, dosimetry estimations showing the highest radiation absorbed doses in the spleen followed by the femur, the skull, the kidneys, the lungs, and the skin draw the attention to the potential organ toxicity of 223 Ra-PLD [121].Therefore, to avoid unwanted radiation exposure, special emphasis must be placed on the protection of healthy organs and tissues-in particular those of the RES system.Although prior studies reported that the administration of isotope-free liposomes prior to the application of the radioactive counterpart appears to decrease the radioactivity of the RES organs and related radiation risk [200], Jonasdottir et al. had contradictory findings, as they experienced the most elevated liposomal radioactivity in the spleen even after pretreatment with unlabeled doxorubicin liposomes (8.1 mg/kg) [121].
In accordance with previous research that confirmed the therapeutic benefit of liposomes loaded with chemotherapeutic drugs [201], we can conclude that alpha emitterlabeled liposomes either with or without anti-tumor agents may have a place in oncological treatment.However, the physicochemical characteristics and the decay characteristics of the labeling isotope should be carefully considered.Compared to other long-lived alpha emitters such as 225 Ac (T 1/2 = 10.0 days) or 224 Ra (T 1/2 = 3.6 days), 223 Ra (T 1/2 = 11.4 days) seems to be a better applicant for liposome-based drug delivery.This is because considering the half-lives of their progenies, in case of 223 Ra the first three, while as for the former two radioisotopes, only the first alpha decay occur in the close proximity of the liposome.It obviously has a major role in the achievement of adequate therapeutic effect as well as in the development of unintended radiation side effects.In addition, to prevent potential leakage from the surface of the liposome and related irradiation of healthy organs/tissues, the development of stable liposomes is another issue of concern.In addition to the integration of biopolymers into the liposome structure [202], the encapsulation of liposomes in nanofibers, hydrogels or films [203,204] also serves to enhance the stability of these nanostructures.Aside from these modifications of liposome preparation, different postprocessing techniques such as freeze drying, spray drying and spray freeze drying appear to be valuable methods for stability enhancement [205].Such postprocessing techniques lead to the development of highly stable dried nanomaterials that could be easily used for drug loading and administration [206].

Other Alpha-Emitting Radionanoprobes for Intraperitoneal Application
In addition to the information detailed above, several other in vivo studies focused on the investigation of ip.nano/microparticle-based therapy with alpha emitters other than 223/224 Ra such as 211 At, 212 Pb, 225 Ac or 227 Th [144,157,207,208].Since the half-life of these isotopes is the major determinant of their peritoneal retention and thus radiotherapeutic effect, the choice of an α-emitter with a suitable lifespan is mandatory for successful antitumor therapy [31].Similarly to 224 Ra-CaCO 3 , 225 Ac, or 227 Th-labeled anti-HER2 antibody trastuzumab induced prolonged survival in in vivo ovarian or ip.tumor models [209][210][211].Although identical anti-tumor effect could be achieved applying comparable amounts of radioactivities of 225 Ac-trastuzmumab, 227 Th-trastuzumab and 224 Ra-CaCO 3 , given the long-lived nature of 225 Ac (T 1/2 : 9.920 (3) days) and 227 Th (T 1/2 : 18.7 days), targeted radiotherapy with 224 Ra (T 1/2 : 3.631 (2) days) seems to be more successful as well as more favorable in terms of radiation safety protection [36,43,212,213].
Comparing the therapeutic efficacy of alpha-emitter 211 At-labeled polymer microspheres ( 211 At-microspheres) to that of 32 P-chromic phosphate colloid or 90 Y-silicate colloid with beta radiation in K13 hybridoma tumor-carrying female Balb/c mice, Vergote et al. reported improved survival and the highest cure rates in the 211 At-treated cohort in comparison with the other two treatment active and the control groups [157].

Highlights for De Novo Nanoradiopharmaceutical Design
Considering the findings of the prior studies on intracavitary treatment with 224 Rananoparticles, we can summarize that the overall therapeutic outcome is determined by several factors including the injected activity, the specific activity, 224 Ra retention, 220 Rn release as well as the re-adsorption of 212 Pb [31,43,132]. 220Rn diffusion may serve to compensate for the downsides of heterogenous microparticle accumulation and related inhomogeneous dose dispersion, while the readsorption of 212 Pb prohibits its delivery to non-target organs.These decay phenomena simultaneously favor the achievement of improved therapeutic effect.Taking these thoughts into account can aid in continuing the establishment of nanosystems capable of a more precise delivery of drug payloads.

Conclusions, Therapeutic Gains form Alpha-Nanoradiopharmaceuticals
Overall, the favorable physicochemical properties of alpha-nanocarriers coupled with their anti-tumor therapeutic benefit confirm the translational potential of alpha nanoplatforms into clinical settings.Targeted radiotherapy with alpha emitters is superior to the other types of radiation in several facets.A reduced incidence of microparticle aggregates in association with alpha therapy ensures a more homogeneous uptake and related uniform dose dispersion compared to other targeted radiotherapeutic treatments, and this in turn favors the preferable application of alpha-radio-nanoparticles [225,226].Macroaggregatesgenerating the self-absorption of the alpha particles into the microparticles as well as off-target tissues and organs-not only reduce the therapeutic effect but also contribute to the development of therapy-associated radiation toxicity.In addition, the five-times-higher relative biological effectiveness (RBE) of alpha radiation compared to beta emission also guarantees the therapeutic success of α-nanoparticles [50].Moreover, due to the short tissue penetration, the use of alpha emitters may yield less toxicity to off-target healthy organs and tissues [227,228].This short tissue range along with high LET values make alpha emitters more successful in the treatment of micrometastatic lesions compared to beta-emitting radionuclides [229].Additionally, alpha therapy ensures sufficient dose delivery to the target site presumably due to the high LET of the alpha particles.
With the enhancement of tumor residence time, nanocarriers have a notable contribution to the augmentation of local radiotoxicity [75,230].Although the application of α-nanotherapy seems to counteract the limitations of β emitters, some downsides of α particles should be carefully considered as well.Given the short penetrating power of α-particles, however, the treatment of sizeable tumor masses brings new challenges and obstacles.Therefore, to ensure therapeutic efficacy, debulking surgery is recommended for tumor mass reduction prior to alpha-radiation [43,231].High production costs as well as the lack of sufficient isotope supply limit the integration of alpha emitters into both preclinical research and clinical applications [232,233].Radiation caused by the daughter radionuclides of the alpha emitters is also a matter of concern and must be handled to impede untoward radiotoxicity of tumor-naïve healthy organs [60].
These days, the focus is centered around imaging diagnostics within the field of nuclear medicine; however, due to the wide availability of alpha and beta emitting isotopes, targeted treatment procedures using radionuclides are demanding more and more space.Owing to the continuous development of the physical technology, radiochemistry, pharmaceutical technology and molecular biology, the number of target-specific alpha-emitting radioisotopes ( 225 Ac, 213 Bi, 224 Ra, 212 Pb, 227 Th, 223 Ra, 211 At, and 149 Tb) is steadily increasing in the preclinical, and hopefully-later on-in the clinical practice as well [38].Beyond the novel and promising radionuclides that are still in their early stage of research, the already well-known and broadly applied Radium isotopes continue to play a key role in one of the newest fields of biotechnology, in nanotechnology.
Overall, the promising results obtained from the studies discussed above demonstrate that the use of nanotechnology in transforming alpha therapy in targeted cancer treatment is the next step forward in today's research.We expect to see more radiolabeled nanoparticles to be established for therapeutic purposes; for the construction of pharmacodynamically optimized candidates, comprehensive future work is required.

Table 1 .
Overview of the preclinical studies on Ra-labeled nanoparticles.

Table 2 .
Toxicity evaluation of the assessed nanoparticles.