Nanomicelles of Radium Dichloride [223Ra]RaCl2 Co-Loaded with Radioactive Gold [198Au]Au Nanoparticles for Targeted Alpha–Beta Radionuclide Therapy of Osteosarcoma

Alpha and beta particulate radiation are used for non-treated neoplasia, due to their ability to reach and remain in tumor sites. Radium-223 (223Ra), an alpha emitter, promotes localized cytotoxic effects, while radioactive gold (198Au), beta-type energy, reduces radiation in the surrounding tissues. Nanotechnology, including several radioactive nanoparticles, can be safely and effectively used in cancer treatment. In this context, this study aims to analyze the antitumoral effects of [223Ra]Ra nanomicelles co-loaded with radioactive gold nanoparticles ([198Au]AuNPs). For this, we synthesize and characterize nanomicelles, as well as analyze some parameters, such as particle size, radioactivity emission, dynamic light scattering, and microscopic atomic force. [223Ra]Ra nanomicelles co-loaded with [198Au]AuNPs, with simultaneous alpha and beta emission, showed no instability, a mean particle size of 296 nm, and a PDI of 0.201 (±0.096). Furthermore, nanomicelles were tested in an in vitro cytotoxicity assay. We observed a significant increase in tumor cell death using combined alpha and beta therapy in the same formulation, compared with these components used alone. Together, these results show, for the first time, an efficient association between alpha and beta therapies, which could become a promising tool in the control of tumor progression.


Introduction
Targeted radionuclide therapy consists of a modality of treatment in which a biological effect is obtained by the energy absorbed from the radiation emitted by the radionuclide.
Firstly, the non-radioactive AuNPs were synthetized. A gold solution of chloroauric acid (HAuCl 4 × 3H 2 O, 0.20 mmol, 78.7 mg) and tetraoctylammonium bromide (TOABr, 0.23 mmol, 126.8 mg) was dissolved in 10 mL of methanol and stirred vigorously for 24 h. Then, sodium borohydride (NaBH 4 2 mmol, 75.6 mg, dissolved in 5 mL of ice water) was added to the mixture, and the solution was kept under stirring for 8 h. After this period, the reaction mixture was ultracentrifuged (40,000 rpm for 30 min) to remove insoluble agglomerates. The supernatant was collected and concentrated by evaporation. The agglomerates were precipitated by adding ethanol to the solution. Then, the precipitate was extracted with minimal amounts of methanol several times. The AuNPs solution was precipitated again by ethanol and finally dried under a vacuum.
Then, the samples of AuNPs were irradiated in the Argonauta reactor (power of 340 W), installed at the Nuclear Engineering Institute (Brazil). The sample was irradiated for 12 h using a thermal neutron flow of 3.2 × 109 n·cm −2 ·s −1 , with an average thermal neutron energy of 0.0025 eV.

Radioactivity Measure
The induced activity of the [ 198 Au]AuNPs was determined by a gamma spectrometry system with a hyper pure germanium (HPGe) detector, with a diameter of 6.2 cm, height of 4 cm, active volume of 41.1 cm 3 , and detection efficiency of 30%, coupled with the multichannel analyzer (Canberra) with 8192 channels. The detector was surrounded by a lead cover of~10 cm to reduce the background. The measurement time for each sample was standardized at 3600 s (1 h).

Detection Efficiency
The detection efficiency for each energy type was determined using a LabSOCS (Laboratory SOurceless Calibration Software, Canberra, Australia). It was necessary to design the geometry used in a computational environment by inserting the physical, chemical, and geometric characteristics of the sample holder used, the detector, and the sample to be analyzed. After entering the data, the software simulates the detection efficiency values for each energy type. Then, the software doubles the number of voxels and repeats the entire process, obeying the convergence criteria and comparing the values until satisfactory convergence is obtained.

Nanomicelles of [ 223 Ra]RaCl 2
A mass of 1 mg/mL of [ 223 Ra]RaCl 2 was weighed and added to the micellar dispersion of Pluronic F127. The system was gently stirred using a magnetic bar (Magnetic Stirrer, IKA, C-MAG HS-7) for 5 min and then processed for 5 min using an ultrasonic processor (UP100H, Hielscher, power: 100%, cycle: 1) in an ice bath at 10 • C.

Nanomicelles of [ 223 Ra]RaCl 2 Co-Loaded with [ 198 Au]AuNPs
A mass of 1 mg/mL of [ 223 Ra]RaCl 2 (~3.7 MBq) was weighed and added to the micellar dispersion of Pluronic F127. The system was gently stirred using a magnetic bar (Magnetic Stirrer, IKA, C-MAG HS-7) for 5 min and then processed for 3 min using an ultrasonic processor (UP100H, Hielscher, power: 100%, cycle: 1) in an ice bath at 10 • C. Then, a mass of 500 µg of [ 198 Au]AuNPs (~1.85 MBq) was added and ultrasonicated for more than 2 min using an ultrasonic processor (UP100H, Hielscher, power: 100%, cycle: 1) in an ice bath at 10 • C. The SaOS-2 cells, a human osteosarcoma cell line, were plated in a density of 1 × 10 4 cells/well for 24h. The cells were maintained in a DMEM/D-glucose (high glucose) medium, supplemented with 10% FBS, penicillin (0.5 U/mL), and streptomycin (0.5 mg/mL). The cells were incubated at 37 • C in a humidified atmosphere of 5% CO 2 . The cells were grown to confluence in 75 cm 2 culture flasks and were detached by brief treatment with trypsin (0.1%)/EDTA (0.01%).

Proliferation Assay
The SaOS-2 cells (1 × 10 4 cells/well) were seeded and allowed to attach for 24 198 Au in the co-loaded nanomicelles is 8.4. After 24 h, the cells were washed, and the number of attached cells was determined using the MTT assay.

Statistical Analysis
Statistical analysis of the data was performed using the GraphPad Prism 7.3 software (GraphPad Software, San Diego, CA, USA). The differences between the means of the two groups were compared using the one-way ANOVA test and confirmed by the Bonferroni post-test. The results are presented as means ± standard deviation (S.D.). The values of * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001 will be considered statistically significant.

Synthesis and Irradiation of Gold Nanoparticles (AuNPs)
Once synthesized, the AuNPs were irradiated under pre-established conditions, and were then able to produce the [ 198 Au]AuNPs. The gamma spectrum obtained with the HPGe detector shows the specific range (412 KeV) of 198 Au, as shown in Figure 1.
constant of 0.4 N/m. However, the actual spring constant was calibrated by the thermal noise method. A drop of all sample solutions was deposited in freshly cleaved mica. The scanning mode used was PeakForce Tapping Quantitative Nanomechanics (QNM), with a resolution of 256 × 256 lines per scan and a scan frequency of 0.5 Hz.

Cell Culture
The SaOS-2 cells, a human osteosarcoma cell line, were plated in a density of 1 × 10 4 cells/well for 24h. The cells were maintained in a DMEM/D-glucose (high glucose) medium, supplemented with 10% FBS, penicillin (0.5 U/mL), and streptomycin (0.5 mg/mL). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells were grown to confluence in 75 cm 2 culture flasks and were detached by brief treatment with trypsin (0.1%)/EDTA (0.01%).

Proliferation Assay
The SaOS-2 cells (1 × 10 4 cells/well) were seeded and allowed to attach for 24 h. The cells were divided into the following three groups:  198 Au in the co-loaded nanomicelles is 8.4. After 24 h, the cells were washed, and the number of attached cells was determined using the MTT assay.

Statistical Analysis
Statistical analysis of the data was performed using the GraphPad Prism 7.3 software (GraphPad Software, San Diego, CA, USA). The differences between the means of the two groups were compared using the one-way ANOVA test and confirmed by the Bonferroni post-test. The results are presented as means ± standard deviation (S.D.). The values of * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001 will be considered statistically significant.

Synthesis and Irradiation of Gold Nanoparticles (AuNPs)
Once synthesized, the AuNPs were irradiated under pre-established conditions, and were then able to produce the [ 198 Au]AuNPs. The gamma spectrum obtained with the HPGe detector shows the specific range (412 KeV) of 198 Au, as shown in Figure 1.

Particle Size
The DLS analysis of [ 198 Au]AuNPs showed the formation of very small nanoparticles (13 nm) with very high monodisperse behavior, confirmed by the PDI (0.106) (Figure 2).

Particle Size
The DLS analysis of [ 198 Au]AuNPs showed the formation of very small nanoparticles (13 nm) with very high monodisperse behavior, confirmed by the PDI (0.106) (Figure 2).

Atomic Force Microscopy
The morphology of the [ 198 Au]AuNPs was investigated by AFM ( Figure 3). Figure 3A shows an AFM height image of a cluster of [ 198 Au] Au nanoparticles. The particles have a homogeneous morphology, with the maximum height on the map reaching 19.7 nm, in regions where the NPs are then superimposed, as evidenced in the three-dimensional image ( Figure 3B). The cross section shown in Figure 3C corresponds to the three NPs marked with a dashed light blue line in Figure 3A. The diameters are 14.4 nm, 10.7 nm, and 13.1 nm, considering the horizontal distance taken from the width at the half-height of the particle. These values follow the DLS results.

Atomic Force Microscopy
The morphology of the [ 198 Au]AuNPs was investigated by AFM ( Figure 3). Figure 3A shows an AFM height image of a cluster of [ 198 Au] Au nanoparticles. The particles have a homogeneous morphology, with the maximum height on the map reaching 19.7 nm, in regions where the NPs are then superimposed, as evidenced in the three-dimensional image ( Figure 3B). The cross section shown in Figure 3C corresponds to the three NPs marked with a dashed light blue line in Figure 3A. The diameters are 14.4 nm, 10.7 nm, and 13.1 nm, considering the horizontal distance taken from the width at the half-height of the particle. These values follow the DLS results.

Particle Size
The dynamic light scattering analysis of [ 223 Ra]RaCl2 nanomicelles showed a mean size of 149 nm, with a PDI of 0.0096 (±0.0002), corroborating the monodispersive value ( Figure 4).   Au]AuNPs. The mean diameter was 296 nm. In addition, it was possible to observe an increase in the PDI value, probably due to the intermicellar destabilization caused by alpha and beta emission, as shown in Figure 5. Au]AuNPs. The mean diameter was 296 nm. In addition, it was possible to observe an increase in the PDI value, probably due to the intermicellar destabilization caused by alpha and beta emission, as shown in Figure 5.

Atomic Force Microscopy of Nanomicelles Systems
The analyses of the ultrastructure of pure nanomicelles (empty), [ 223 Ra]RaCl 2 nanomicelles, and [ 223 Ra]RaCl 2 co-loaded with [ 198 Au]AuNPs nanomicelles were performed by AFM, and compared with the 127-Pluronic blank nanomicelles sample ( Figure 6). Figure 6A shows a 127-Pluronic white nanomicelles film. Polymeric chain structures, with a diameter of 263.4 ± 12.1 nm, are observed. The maximum film height is 359 nm. The threedimensional representation of Figure 6 is shown in Figure 6D-F, in which the absence of globular structures is evident.

In Vitro Cytotoxicity
The cytotoxicity effect on human osteosarcoma of the nanosystems developed is expressed in Figure Figure 6F). Since these holes are only observed in the 127-Pluronic-[ 223 Ra] and [ 223 Ra]RaCl 2 co-loaded with [ 198 Au]AuNPs films, they can be promoted by emitting alpha and beta particles from the radioactive nanomicelles. However, this emission is not able to destabilize the nanomicelles clusters.

In Vitro Cytotoxicity
The cytotoxicity effect on human osteosarcoma of the nanosystems developed is expressed in Figure 7. It is possible to observe a very potent effect, mainly in 127-Pluronic-

Discussion
Radionuclide therapy is a safe and effective approach to treat primary cancers, as well as distant metastases. In this sense, beta-or alpha-emitting radionuclides have been primarily used [70]. However, there are a lack of radiopharmaceuticals that deliver simultaneous alpha and beta radiations, which would have remarkable potential for treating tumors. Alpha and beta particles have high energy in different magnitudes; consequently, they have different mean penetration ranges in tissue, for depositing their energies. Hence, alpha-/beta-labeled radiopharmaceuticals would combine the long-range crossfire effect of beta radiation with the DNA localization effect of alpha radiation, among other effects [1]. In addition, successful treatment would be reached using lower doses of alpha and beta emitters. Therefore, we designed and prepared a novel nanoradiopharmaceutical, containing [ 223 Ra]RaCl2 co-loaded with [ 198 Au]AuNPs into nanomicelles, for radionuclide therapy of bone cancer using alpha and beta radiations simultaneously. Here, to the best of our knowledge, we presented the first findings of combining alpha and beta therapy in the same formulation. In addition, we are the first to report the radiolabeling of nanomicelles with 223 Ra for delivering alpha radiation into tumor cells.
To fulfill our goal, the non-radioactive AuNPs were firstly fabricated using chemical synthesis. Through light scattering analysis, the AuNPs obtained a mean size of 13 nm and a PDI value of 0.106 (±0.089). Furthermore, the AFM analyses corroborated the DLS results, confirming the quality and homogeneity of our AuNPs. Then, the AuNPs were activated under neutron irradiation to obtain the [ 198 Au]AuNPs, similar to previous reports [11]. Biodistribution studies (Table S1-

Discussion
Radionuclide therapy is a safe and effective approach to treat primary cancers, as well as distant metastases. In this sense, beta-or alpha-emitting radionuclides have been primarily used [70]. However, there are a lack of radiopharmaceuticals that deliver simultaneous alpha and beta radiations, which would have remarkable potential for treating tumors. Alpha and beta particles have high energy in different magnitudes; consequently, they have different mean penetration ranges in tissue, for depositing their energies. Hence, alpha-/beta-labeled radiopharmaceuticals would combine the long-range crossfire effect of beta radiation with the DNA localization effect of alpha radiation, among other effects [1]. In addition, successful treatment would be reached using lower doses of alpha and beta emitters. Therefore, we designed and prepared a novel nanoradiopharmaceutical, containing [ 223 Ra]RaCl 2 co-loaded with [ 198 Au]AuNPs into nanomicelles, for radionuclide therapy of bone cancer using alpha and beta radiations simultaneously. Here, to the best of our knowledge, we presented the first findings of combining alpha and beta therapy in the same formulation. In addition, we are the first to report the radiolabeling of nanomicelles with 223 Ra for delivering alpha radiation into tumor cells.
To fulfill our goal, the non-radioactive AuNPs were firstly fabricated using chemical synthesis. Through light scattering analysis, the AuNPs obtained a mean size of 13 nm and a PDI value of 0.106 (±0.089). Furthermore, the AFM analyses corroborated the DLS results, confirming the quality and homogeneity of our AuNPs. Then, the AuNPs were activated under neutron irradiation to obtain the [ 198 Au]AuNPs, similar to previous reports [11]. Biodistribution studies ( Hence, these findings are very promising. Radionuclide therapy has the advantage of delivering a concentrated dose to target tumor tissues, while preserving the surrounding healthy tissues, unlike the other current cancer therapies [19,39,67,71,72]. For the first time, the simultaneous ability of alpha and beta radiations to kill cancer cells has been demonstrated, using a low radioactive dose and obtaining high efficacy.

Conclusions
The combination of alpha and beta radiations in the same nanoprobe would represent a very efficient tool for cancer treatment. Our in vitro findings are the first to demonstrate the remarkable ability of alpha-beta nanoprobes to kill cancer cells using a low radioactive dose. Future works should aim to evaluate the in vivo therapeutic effect and safe use of our nanomicelles of [ 223 Ra]RaCl 2 co-loaded with [ 198 Au]AuNPs in bone cancer-bearing mice. Conversely, this work may lead to further studies involving the functionalization of these alpha-beta nanoprobes for targeted radionuclide therapy beyond bone cancer.