Astatine-211-Labeled Gold Nanoparticles for Targeted Alpha-particle Therapy via Intravenous Injection

doi:10.21203/rs.3.rs-2110991/v1

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Astatine-211-Labeled Gold Nanoparticles for Targeted Alpha-particle Therapy via Intravenous Injection

https://doi.org/10.21203/rs.3.rs-2110991/v1

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Background:

Alpha-particle radiotherapy has gained considerable attention owing to its potent anti-cancer effect. ²¹¹At, with a relatively short half-life of 7.2 h, emits an alpha particle within a few cell diameters with high kinetic energy, which damages cancer cells with high biological effectiveness. In this study, we investigated the intravenous injection of ²¹¹At-labeled gold nanoparticles (AuNPs) for targeted alpha-particle therapy (TAT).

Results:

Different kinds of surface-modified gold nanoparticles can be labeled with ²¹¹At in high radiochemical yield in 5 min, and no purification is necessary. In vivo biodistribution results showed the accumulation of 5 nm ²¹¹At-AuNPs@mPEG at 2.25 % injection dose per gram in tumors within 3 h via the enhanced permeability and retention effect. Additionally, we observed a long retention time in tumor tissues within 24 h. This is the first study to demonstrate the anti-tumor efficacy of 5 nm ²¹¹At-AuNPs@mPEG that can significantly suppress tumor growth in a pancreatic cancer model via intravenous administration.

Conclusions:

AuNPs are satisfactory carriers for ²¹¹At delivery, due to simple and efficient synthesis processes and high stability. Intravenous administration of 5 nm ²¹¹At-AuNPs@mPEG has a significant anti-tumor effect. This study provides a new framework for designing nanoparticles suitable for targeted alpha-particle therapy via intravenous injection.

Astatine-211

Gold nanoparticles

Targeted alpha-particle therapy

Cancer therapy

Pancreatic cancer

Intravenous administration

Radionuclide cancer therapeutics are pharmaceuticals that use short-lived radionuclides that emit α or β particles to kill cancer cells via intracellular or intratumoral radiation. Recently, much attention has been given to the potent cancer therapeutic efficacy of targeted alpha-particle therapy (TAT) (radionuclides that emit α particles) [1, 2]. Compared to β particles, α particles possess higher kinetic energy and ionization power. Therefore, TAT can significantly induce DNA double-strand breaks (DSB) that effectively damages cancer cells, while damage to normal cells damage is limited owing to the short penetration range. Considering the decay process, half-life, and manufacturing processes of radionuclides, there are a limited number of α-emitting radionuclides suitable for TAT, such as ²²³Ra, ²²⁵Ac, and ²¹¹At [2]. So far, ²²³Ra dichloride is the only TAT drug approved by the U.S. Food and Drug Administration and European Medicines Agency for treating patients with castration-resistant prostate cancer, and symptomatic bone metastases [3]. ²²³Ra mimics calcium and forms complexes with hydroxyapatite in areas with increased bone turnover. Thus, selective accumulation of radio-pharmaceuticals is vital for TAT to effectively suppress adverse effects on normal cells. Generally, TAT drugs are delivered to cancer cells by binding radionuclides to carrier (vehicle) drugs that target cancers. Radionuclide ²²⁵Ac, which has been extensively studied for cancer treatments, can be attached to molecular targeted drugs via chelate formation. For instance, ²²⁵Ac-PSMA-617 was of particular interest owing to its therapeutic effect on tumors that metastasized throughout the body. After ²²⁵Ac-PSMA-617 treatment, the majority of the tumor seemed to disappear, and prostate-specific antigen tests further indicated that metastasized prostate cancer was cured to a large extent [4]. Additionally, a study at Osaka university also showed that ²²⁵Ac-labeled fibroblast activation protein inhibitors (FAPI) could be used to treat fibroblast activation protein(FAP)-expressing pancreatic cancer [5]. Globally, especially in Japan, another kind of α particles emitting radionuclides, astatine-211(²¹¹At), has attracted considerable attention and multiple ²¹¹At labeled compounds are currently under development [6, 7].

Astatine is one of elements in the halogens; thus, it shares some chemical properties with iodine. Sodium-iodide symporter plays a significant role in the accumulation of iodine as well as astatine [8]. Taking advantage of this property, a research team led by Osaka University, used sodium astatide (²¹¹At-NaAt) to suppress refractory thyroid cancer [9]. Adding ascorbic acid to the ²¹¹At solution can significantly increase the radiochemical purity of astatide and enhances uptake by the thyroid in rats. Furthermore, in a murine xenograft model, the mice subjected to 1 MBq of ²¹¹At-NaAt survived nearly three times as long as untreated mice, and their tumors were several-fold times smaller. Currently clinical research on cancer treatment using ²¹¹At-NaAt is being actively promoted at Osaka University [10].

Unlike ²²⁵Ac, ²¹¹At can be introduced to carriers via covalent bonding. Hence, small-molecule drugs targeting cancer cells can be used as carrier. Kaneda-Nakashima et al. in Osaka University prepared ²¹¹At-labeled α-methyl-L-tyrosine (²¹¹At-AAMT) targeting a cancer-specific L-type amino acid transporter 1 (LAT1) which is highly expressed in cancer cells but rarely expressed in normal cells [11]. ²¹¹At-AAMT exhibited a high affinity for LAT1, inhibited tumor cells, and caused DNA DSBs in vitro. ²¹¹At-AAMT was selectively accumulated in tumors and had remarkable anti-cancer effects in a murine tumor model [12].

Our research explored the substantial potential of gold nanoparticles (AuNPs) as the carrier for the targeted delivery of ²¹¹At via intravenous administration. In a previous study, we found that ²¹¹At was efficiently introduced to AuNPs by mixing ²¹¹At with AuNPs solution for 5 min with a high radiochemical yield (RCY), and no purification process was necessary [13]. Since AuNPs facilitate a variety of surface modifications and have been widely used as drug carriers for diagnostic or therapeutic agents [14–17], we investigated the applications of ²¹¹At-labeled gold nanoparticles (²¹¹At-AuNPs) for TAT. We firstly showed that intratumoral administration of ²¹¹At-AuNPs can strongly suppress tumor growth, and also showed the stability of ²¹¹At-AuNPs in the body [13]. Although some studies have shown the in vitro and in vivo studies of ²¹¹At-AuNPs [13, 18, 19], the anti-tumor effect of ²¹¹At-AuNPs via intravenous injection has not been previously investigated.

Here, four kinds of functional AuNPs were investigated. These can be divided into two groups: nanoparticles modified with mPEG (5 nm and 30 nm AuNPs@mPEG) and nanoparticles modified with peptides and mPEG (5 nm AuNPs@H16 and AuNPs@H16/RGD). The nanoparticles modified with mPEG were expected to accumulate in tumor tissues via the enhanced permeability and retention (EPR) effect. Since the half-life of ²¹¹At is short, the rapid accumulation of ²¹¹At-labeled compounds in cancer cells has a pivotal role in alpha therapy with minimal side effects on normal tissues. We took advantage of the EPR effect and the fact that cancer cells have a higher nanoparticle uptake rate than normal cells to develop a novel TAT drug.

Additionally, we investigated the polyhistidine peptide (H16) modified gold nanoparticles (5 nm AuNPs@H16) and polyhistidine peptide (H16) and cyclic RGD peptide-modified gold nanoparticles (AuNPs@H16/RGD). The H16 peptide [20] and cyclic RGD [21, 22] can target tumor pH and the integrin αvβ3, respectively, peptide modification can also promote cellular uptake of nanoparticles. The integrin αvβ3 is highly expressed on tumor cells and new-born vessels between tumor tissue. Many human pancreatic cancer cells, including PANC-1 cell line used in this research, were reported for its high expression of integrin αvβ3 [23]. Furthermore, solid tumor tissues also showed low pH condition, which can be used as an appropriate target [20].

Astatine-211 production

²¹¹At was produced in the ²⁰⁹Bi(α,2n)²¹¹At reaction using the RIKEN AVF Cyclotron and purified as previously described [9, 13, 24].

AuNPs modification and characterization

AuNPs (5 nm and 30 nm) (OD 1, stabilized suspension in citrate buffer) were purchased from Sigma-Aldrich. For mPEG modification, mPEG thiol (Mn 6,000) was added to aqueous solutions containing AuNPs of 5 nm and 30 nm so that the final concentration of mPEG thiol was 0.1 M. The reaction mixtures were stirred at room temperature for 2 h. After that, the mPEG-modified AuNPs were purified by centrifugation as we reported in previous study in order to obtain 5 nm and 30 nm AuNPs@mPEG. H16 peptide (2-mercaptopropionic acid-HHHHH HHHHH HHHHH H-NH₂ trifluoroacetate salt) and RGD peptide (cyclo[Arg-Gly-Asp-D-Phe-Lys(Cys)]) were purchased from Peptide Institute. Inc. (Ibaraki, Osaka, Japan). For H16 and RGD peptide modifications, we first added the mPEG thiol (Mn 2000) to the 5 nm AuNPs solutions, and the mixtures were stirred at room temperature for 10 min. The mixtures were centrifuged once, and the solvent was changed to 0.1 M phosphate buffer (pH 6.0). Next, the H16 peptide solution or H16 and RGD peptide mixture solution was added to the mPEG-modified AuNPs dissolved in phosphate buffer (pH 6.0), and the solutions were stirred for 3 h at room temperature. After the reaction, the H16-modified AuNPs or H16 and RGD double-modified AuNPs were purified centrifuged by using Amicon® ultra-0.5 centrifugal filter devices (50K) at 10000 × g for 5 min. Following centrifugation, 0.1 M phosphate buffer (pH 6.0) was added to the devices. This operation was performed twice to obtain 5 nm AuNPs@H16 or AuNPs@H16/RGD. AuNPs@mPEG (120 nm) was prepared as previously described [13]. The quality of the synthesized AuNPs was confirmed by transmission electron microscopy (TEM) (JEM-2100; JEOL Ltd., Tokyo, Japan). Dynamic light scattering (DLS) and zeta potential measurements of surface-modified AuNPs were measured by using Zetasizer Ultra (Malvern Panalytical Ltd., Malvern, UK). The ultraviolet-visible (UV-Vis) absorption spectrum of modified AuNPs was determined using a UV-Visible/NIR spectrophotometer (V-730; JASCO Corp., Tokyo, Japan).

²¹¹ At Radiolabeling yield evaluation

²¹¹At aqueous solution (30 µL, 4.5—6.0 MBq) was added to 70 µL of modified AuNPs aqueous solution, and the mixture was shaken at room temperature for 5 min. The radioactivity of ²¹¹At-AuNPs was then measured by a germanium semiconductor detector BE-2020; Mirion Technologies (Canberra), Inc., Meriden, CT, USA). Then, the solution was transferred into 50K Amicon® ultra centrifugal filters and separated by centrifugal filtration (6000 × g, 10 min) for twice. Through radioactivity measurement of reaction tubes, filtrates, filter devices and tips, we could calculate the RCY of the astatination reaction. Radioactive decay correction was done according to the measurement time.

In vivo biodistribution and therapy efficacy

The experimental protocol was approved by the Animal Care and Use Committee of the Osaka University Graduate School of Science. All animals were housed under a 12-h dark–light cycle (light from 08:00 to 20:00) at 25 ± 1 ° C with ad libitum food (CRF-1: Oriental Yeas Co., Ltd. Tokyo, JAPAN) and water in a multi-chamber animal housing System (Nippon Medical & Chemical Instruments. Co., Ltd. Osaka, Japan). BALB/c nu/nu mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). PANC-1 cells were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% antibiotics in a humidified incubator with 5% CO₂. Cultured cells were washed in phosphate-buffered saline (PBS) and harvested with trypsin.

Tumor xenograft models were established by subcutaneous injection of 1×10⁷ cells in 0.2 mL of serum-free medium and Matrigel (1:1; BD Bioscience, Franklin Lakes, NJ, USA) into male BALB/c-nu/nu mice.

For the distribution assay, PANC-1 mice (n = 32; twelve weeks old; male body weight = 24.4 ± 1.1 g) were used for evaluation at 3 h and 24 h following administration of the 5 nm ²¹¹At-AuNPs@mPEG (241.5 ± 22.3 kBq/mouse), 30 nm ²¹¹At-AuNPs@mPEG (211.4 ± 3.4 kBq/mouse), ²¹¹At-AuNPs@H16 (405.7 ± 11.4 kBq/mouse), ²¹¹At-AuNPs@H16 H16/RGD (410.3 ± 10.2 kBq/mouse). All organs were packed into a zippered polyethylene bag and radioactivity was measured with a gamma counter (2480 Wizard2, PerkinElmer).

For the therapeutic assay, PANC-1 xenograft mice (n = 12, 7 weeks old; body weight = 23.3 ± 1.1 g) were used when tumor size reached approximately 50 mm³ on average. The mice were divided into three groups according to the injected dose [1 MBq (989.2 ± 6.3 kBq/mouse, n = 4), 0.5 MBq (515.9 ± 8.6 kBq/mouse, n = 4), and control (n = 4)]. The control group received no ²¹¹At labeled AuNPs. Tumor sizes and body weights were measured thrice per week. The mice were euthanized when the tumor size reached more than 10% of the total weight. The mice were followed for 30 days.

Internalization evaluation

PANC-1, MDA-MB-231, Hs 578Bst cells were cultured at 37°C in D-MEM containing 10% fetal bovine serum and 1% antibiotics in a humidified incubator with 5% CO₂. The cultured cells were washed in PBS and harvested with trypsin.

The internalization of AuNPs (no radionuclide) were evaluated using reflectance imaging as we reported in previous study [13]. Briefly, PANC-1 cells (1×10⁴ cells/well in 100 µL of medium), MDA-MB-231 cells (5×10⁴ cells/well in 100 µL of medium), and Hs 578Bst cells (5×10⁴ cells/well in 100 µL of medium) were seeded on to 96-well plates (glass bottom) and treated with the different kinds of AuNPs for 24 h. The cells were then washed, and fixed with 4% paraformaldehyde solution, and the cell nucleus was stained by Hoechst 33342. Micorgraphs were taken with a Nikon A1R + inverted confocal microscope (Nikon Corp., Tokyo, Japan) and a Plan Apo VC water-immersion objective lens (60 ×, NA = 1.20; Nikon Corp., Tokyo, Japan).

The internalization of ²¹¹At labeled AuNPs was quantitated by determination of the radioactivity. Briefly, the PANC-1 cells were seeded in 96-well plates and the different kinds of AuNPs solutions were added to each well. Each sample was added to the cells and incubated for 5 h. After treatment, the cells were washed twice with 1X PBS, and lysed with 1N NaOH. The radioactivity levels in the lysed cell samples were measured with a gamma counter, 2480 Wizard² (PerkinElmer, Inc.). The protein levels were determined with a BCA protein assay kit (Thermo Fisher Scientific).

Statistical analysis

The P-value was calculated with one-way ANOVA using GraphPad Prism software. P-values < 0.05 were considered statistically significant.

²¹¹ At labeled AuNPs synthesis

Four kinds of ²¹¹At labeled functional AuNPs (5 nm ²¹¹At-AuNPs@mPEG, 30 nm ²¹¹At-AuNPs@mPEG, 5 nm ²¹¹At-AuNPs@H16, and 5 nm ²¹¹At-AuNPs@H16/RGD) were designed as shown in Fig. 1. The synthesized surface-modified AuNPs were evaluated via TEM, DLS, and UV-Vis, and the results are shown in Figure S1 and Table S1. All kinds of AuNPs were found to be approximately spherical and reasonably monodispersed. The different surface modifications affected their zeta-potentials. The mPEG modified AuNPs were well dispersed in an aqueous solution. While the two kinds of peptides modified AuNPs were aggregated in water as the solvent during the modification process, they were dispersed and stable in PB solution (pH 6.0). To evaluate the stability of 5 nm ²¹¹At-AuNPs@H16 and ²¹¹At-AuNPs@H16/RGD, samples were diluted in phosphate buffer (pH7.4 or 8.0), and DLS measurement was performed. The two kinds of peptides modified AuNPs were reasonably monodispersed and no notable size change was observed (Table S2).

After 5 min of mixing the AuNPs and ²¹¹At solution, the ²¹¹At labeling efficiency was evaluated as previously describe. The labeling conditions and RCY are shown in Table S2 and Figure S2. Although there was some adhesion to the reaction tube walls and pipette tips, the free ²¹¹At in the filtrate was less than 1%, and all final RCYs were above 90%.

Biodistribution study

Four kinds of ²¹¹At-AuNPs were intravenously injected into mice, and after 3 h or 24 h, the radioactivity of organ tissues was measured to evaluate the biodistribution. The biodistribution (%ID/g and %ID) of 5 nm and 30 nm ²¹¹At-AuNPs@mPEG is shown in Table 1 and Table 3. The level of 5 nm ²¹¹At-AuNPs@mPEG accumulated in tumor tissue at 3 h post-administration was 2.25 ± 0.67% ID/g, which was higher than that of 30 nm ²¹¹At-AuNPs@mPEG (1.29 ± 0.17% ID/g). After 24 h, the accumulation of AuNPs could still be observed in tumor tissues, which were 1.80 ± 0.20% ID/g and 0.85 ± 0.21% ID/g for 5 nm and 30 nm ²¹¹At-AuNPs@mPEG, respectively. The data obtained from urine, indicated rapid clearance between 3 and 24 h post-administration.

Table 1

Comparison of percentage distribution of injected dose per gram (% ID/g) of 5 nm and 30 nm ²¹¹At-AuNPs@mPEG
	3 h		24 h
	5 nm ²¹¹At-AuNPs@mPEG	30 nm 211At-AuNPs@mPEG	5 nm ²¹¹At-AuNPs@mPEG	30 nm ²¹¹At-AuNPs@mPEG
Thyroid	12.12 ± 5.69	2.61 ± 0.39	4.560 ± 1.05	6.98 ± 2.33
Liver	2.88 ± 0.82	5.87 ± 3.79	2.18 ± 0.37	2.08 ± 1.16
Stomach	11.73 ± 5.22	2.03 ± 0.56	2.45 ± 0.60	4.71 ± 2.19
Small intestine	2.10 ± 0.76	0.85 ± 0.13	0.81 ± 0.09	0.74 ± 0.23
Colon	1.36 ± 0.41^#	0.34 ± 0.02	0.88 ± 0.18	0.67 ± 0.24
Kidney	2.80 ± 0.58^#	1.27 ± 0.21	1.90 ± 0.26	1.17 ± 0.38
Blood	7.06 ± 0.88^*###	0.59 ± 0.04	2.60 ± 0.54^#	0.57 ± 0.20
Urine	8.99 ± 3.23	3.73 ± 1.67	14.68 ± 1.97^#	14.90 ± 3.72
Tumor	2.25 ± 0.67	1.29 ± 0.17	1.80 ± 0.20^#	0.85 ± 0.21
Mean ± standard error (S.E.) of % ID/g is shown in the table. Significant difference between 3 and 24 h is indicated as ^p < 0.05 and ^*p < 0.01, and between 5 nm and 30 nm at 24 h as ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001.

The biodistribution (%ID/g and %ID) of 5 nm ²¹¹At-AuNPs@H16 and ²¹¹At-AuNPs@H16/RGD is shown in Table 2 and Table 4. At 3 h after the injection, the accumulation of ²¹¹At-AuNPs@H16 and ²¹¹At-AuNPs@H16/RGD was high in the liver (Table 2 and Table 4), i.e., 17.83 ± 8.54% ID/g and 16.02 ± 7.08% ID/g, respectively. A higher accumulation of RGD peptides modified AuNPs (41.59 ± 2.20% ID/g) in the liver 24 h post-administration was observed. However, in tumor tissues, 1.36 ± 0.44% ID/g and 2.34 ± 0.94% ID/g of 5 nm ²¹¹At-AuNPs@H16 and 5 nm ²¹¹At-AuNPs@H16/RGD, respectively, were accumulated at 3 h post-administration. After 24 h, 1.31 ± 0.27% ID/g of ²¹¹At-AuNPs@H16 and 2.07 ± 0.47% ID/g of ²¹¹At-AuNPs@H16/RGD remained in the tumor tissue. Compared to mPEG-modified AuNPs, tumor enrichment of AuNPs was not significantly improved by peptide modification.

Table 2

Comparison of percentage distribution of injected dose per gram (% ID/g) of ²¹¹At-AuNPs by peptide modification.
	3 h		24 h
	5 nm ²¹¹At-AuNPs@H16	5 nm ²¹¹At-AuNPs@H16/RGD	5 nm ²¹¹At-AuNPs@H16	5 nm ²¹¹At-AuNPs@H16/RGD
Thyroid	3.64 ± 1.38	3.29 ± 1.29	3.34 ± 0.47	2.60 ± 0.51
Liver	17.83 ± 8.54	16.02 ± 7.08^*	16.91 ± 6.48	41.59 ± 2.20^#
Stomach	3.74 ± 1.19	3.89 ± 1.23	1.73 ± 0.20	2.45 ± 0.56
Small intestine	0.85 ± 0.28	1.11 ± 0.31	0.46 ± 0.06	1.01 ± 0.07^###
Colon	0.68 ± 0.19	0.67 ± 0.15	0.43 ± 0.11	0.72 ± 0.10
Kidney	1.74 ± 0.46	1.64 ± 0.56	0.85 ± 0.12	2.09 ± 0.28^###
Blood	6.91 ± 1.48^**	7.44 ± 1.59^**	0.64 ± 0.11	0.44 ± 0.05
Urine	12.98 ± 5.97	8.04 ± 1.79^**	4.17 ± 1.29	0.44 ± 0.43^#
Tumor	1.36 ± 0.44	2.34 ± 0.94	1.31 ± 0.27	2.07 ± 0.47
Mean ± standard error (S.E.) of % ID/g is shown in the table. Significantly difference between 3 and 24 h is indicated as ^p < 0.05 and ^*p < 0.01, and between 5 nm ²¹¹At-AuNPs@H16 and 5 nm ²¹¹At-AuNPs@H16/RGD in 24 h as ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001.

Table 3

Comparison of percentage distribution of injected dose (% ID) of 5 nm and 30 nm ²¹¹At-AuNPs@mPEG.
	3 h		24 h
	5 nm ²¹¹At-AuNPs@mPEG	30 nm 211At-AuNPs@mPEG	5 nm ²¹¹At-AuNPs@mPEG	30 nm ²¹¹At-AuNPs@mPEG
Thyroid	0.67 ± 0.26	0.19 ± 0.02	0.31 ± 0.05	0.42 ± 0.12
Liver	3.97 ± 1.17	7.61 ± 5.22	2.68 ± 0.41	2.70 ± 1.42
Stomach	3.96 ± 2.00	0.52 ± 0.17	0.64 ± 0.19	1.23 ± 0.51
Small intestine	1.93 ± 0.73	0.82 ± 0.12	0.86 ± 0.10	0.81 ± 0.18
Colon	0.70 ± 0.39	0.08 ± 0.02	0.23 ± 0.06	0.14 ± 0.03
Kidney	1.23 ± 0.25^#	0.55 ± 0.08	0.81 ± 0.10	0.48 ± 0.14
Blood	1.79 ± 0.43^#	0.30 ± 0.09	1.26 ± 0.20	0.29 ± 0.07^##
Urine	0.57 ± 0.22	0.63 ± 0.36	2.24 ± 0.58^*	0.14 ± 0.11^#
Tumor	2.03 ± 0.62	1.45 ± 0.21	1.92 ± 0.77	0.91 ± 0.44
Mean ± standard error (S.E.) of % ID is shown in the table. Significantly difference between 3 and 24h is indicated as ^*p < 0.05, and between 5 nm and 30 nm in 24 h as ^#p < 0.05 and ^##p < 0.01.

Table 4

Comparison of percentage distribution injected dose (% ID) of ²¹¹At-AuNPs by peptide modification.
	3 h		24 h
	5 nm ²¹¹At-AuNPs@H16	5 nm ²¹¹At-AuNPs@H16/RGD	5 nm ²¹¹At-AuNPs@H16	5 nm ²¹¹At-AuNPs@H16/RGD
Thyroid	0.23 ± 0.09	0.17 ± 0.06	0.17 ± 0.02	0.15 ± 0.05
Liver	23.32 ± 10.85	20.73 ± 8.52	22.09 ± 7.91	44.26 ± 1.78^*#
Stomach	1.45 ± 0.41	1.54 ± 0.46	0.66 ± 0.10	0.83 ± 0.15
Small intestine	1.18 ± 0.39	1.34 ± 0.39	0.53 ± 0.08	1.08 ± 0.11^##
Colon	0.13 ± 0.04	0.19 ± 0.07	0.14 ± 0.06	0.17 ± 0.01
Kidney	0.69 ± 0.18	0.67 ± 0.26	0.34 ± 0.04	0.69 ± 0.10^#
Blood	2.91 ± 0.41	3.79 ± 0.64	0.25 ± 0.03^***	0.23 ± 0.03^**
Urine	1.27 ± 0.85	0.42 ± 0.16	0.09 ± 0.03	0.53 ± 0.17^#
Tumor	0.28 ± 0.14	0.45 ± 0.19	0.23 ± 0.09	0.15 ± 0.03
Mean ± standard error (S.E.) of % ID is shown in the table. Significantly difference between 3 and 24 h is indicated as ^p < 0.05, ^p < 0.01, ^**p < 0.001, and between 5 nm ²¹¹At-AuNPs@H16 and 5 nm ²¹¹At-AuNPs@H16/RGD in 24 h ^#p < 0.05, ^##p < 0.01.

In vivo therapeutic effect

Considering the higher accumulation of ²¹¹At-AuNPs@H16 and ²¹¹At-AuNPs@H16/RGD in the liver, we assume these have a stronger side effect and that they may not be suitable for intravenous administration. Since 5 nm ²¹¹At-AuNPs@mPEG showed relatively higher accumulation in tumor tissues and relatively lower accumulation in other organs, we ultimately used 5 nm ²¹¹At-AuNPs@mPEG to investigate the in vivo therapeutic effect.

After administration, no inflammation or abnormalities were observed around the injection site. The tumor size change after administration of 5 nm ²¹¹At-AuNPs@mPEG (1 MBq/mL or 0.5 MBq/mL) and AuNPs@mPEG (0 MBq/mL) are shown in Fig. 2A. After one month, the mice were euthanized, and the tumors were removed to measure their weight, as shown in Figs. 2B and C. Tumor proliferation was significantly inhibited by treatment with ²¹¹At-AuNPs@mPEG in the 1 MBq and 0.5 MBq groups. Although %ID in the tumors was 2.03 ± 0.27%ID (3 h) and 1.92 ± 0.77%ID (24 h), the tumor growth was suppressed by treatment with ²¹¹At-AuNPs. The mouse body weight slight decreased after administration of ²¹¹At-AuNPs@mPEG but recovered after 2 weeks, as shown in Fig. 2D.

Cellular uptake of AuNPs and DNA DSBs in vitro

The cellular uptake of AuNPs without ²¹¹At labeling was investigated using reflectance imaging. Cold AuNPs were added to the cell and incubated for 24 h. As shown in Figure S3A, 5 nm AuNPs@H16 and AuNPs@H16/RGD were accumulated in the cells, while the uptake of 5 nm and 30 nm mPEG modified AuNPs was quite low in vitro.

The internalization of 120 nm AuNPs@mPEG in the breast cancer (MDA-MB-231) and normal mammary gland tissue (Hs 578Bst) cell lines were compared (Figure S5). Many visible black AuNP aggregates were observed in the MDA-MB-231 cells, whereas in the Hs 578Bst cell line, AuNP uptake was low and AuNPs were evenly distributed within the cells.

In addition, the cellular uptake of ²¹¹At-AuNPs was quantified by determining the radioactivity of PANC-1 cell lysates as shown in Figure S3B. After 5 h of incubating the cells with ²¹¹At-AuNPs, the internalization level of the two kinds of mPEG modified AuNPs was low, whereas the internalization of the two kinds of peptides modified AuNPs was significantly higher than that of mPEG modified AuNPs.

γH2AX was used as the marker of DNA DSB induction. The results of DSB induction are shown in Figure S4. ²¹¹At-AuNPs@mPEG (5 nm and 30 nm) induced low levels of DSB induction. The two kinds of peptides-modified AuNPs caused a higher DNA DSB induction after 5 h of incubation with 1 MBq/mL of ²¹¹At-AuNPs@H16 or ²¹¹At-AuNPs@H16/RGD in a concentration-dependent manner.

Gold nanoparticles have been used as a delivery carrier for ²¹¹At due to the strong affinity of astatine to gold [13, 18, 19, 25, 26]. We recently reported that ²¹¹At-AuNPs@mPEG exhibited cytotoxicity when they were absorbed by the tumor cells and the intratumoral administration of ²¹¹At-AuNPs@mPEG strongly suppressed the cancer growth in a particle size-dependent manner. Smaller particles showed a more rapid distribution in tumor regions, and 5 nm ²¹¹At-AuNPs@mPEG showed the highest suppression of cancer growth among various sizes of AuNPs tested [13]. A similar nanoseed brachytherapy using ²¹¹At-labeled gold nanostars has also reported significant inhibition of the growth of human gliomas in a murine model [25].

Here, we demonstrated that 5 nm ²¹¹At-AuNPs@mPEG significantly suppressed tumor growth in a pancreatic cancer model via intravenous administration. Accumulation of ²¹¹At-AuNPs@mPEG in PANC-1 tumors occurred via passive targeting through the EPR effect. The 5 nm ²¹¹At-AuNPs@mPEG showed a higher %ID of accumulation in tumors than 30 nm ²¹¹At-AuNPs@mPEG, suggesting the higher vascular permeability and the higher diffusion into tumor tissue. Many factors affect the EPR effect of AuNPs, including the size, surface modification of nanoparticles and tumor microenvironment [27–31]. Sykes et al. reported that passively targeted AuNPs with diameters less than 45 nm (core size, hydrodynamic diameter 78 nm) easily permeate the tumor [32]. Liu et al. reported that 30 nm gold nanostars have higher tumor uptake and deeper penetration into tumor interstitial space than their 60 nm counterparts [33].

In contrast, in vitro cellular uptake and DNA DSB induction of both mPEG-modified AuNPs were low. We also report that in vitro uptake of ²¹¹At-labeled AuNPs does not correlate with their in vivo anticancer effects in our previous intratumoral administration study [13]. The 120 nm ²¹¹At-AuNPs@mPEG showed evident cytotoxicity in an in vitro cellular assay, while ²¹¹At-labeled AuNPs of smaller sizes did not show cytotoxicity. The 120 nm AuNPs were precipitated by gravity, allowing the AuNPs to contact cells, and the uptake of the AuNPs into the cells led to cell death. The other AuNPs were dispersed in solution and were not taken up by the cells in the in vitro system, therefore no cytotoxicity was observed. In contrast, the intratumoral administration of 5 nm ²¹¹At-AuNPs@mPEG exhibited the most potent anticancer activity. These results indicate that 5 nm AuNPs are efficiently dispersed in tumor tissue and rapidly internalized into cancer cells in vivo by both intravenous and intratumoral administrations.

Significant adverse effects were not observed following the intravenous administration of 5 nm ²¹¹At-AuNPs@mPEG, although a slight weight loss was observed in the early phase. The difference in uptake of AuNPs between cancer cells and normal cells can explain the low toxicity of 5 nm ²¹¹At-AuNPs@mPEG to normal cells. The “eating” ability of cancer cells should be much higher than that of normal cells, since tumors require abundant nutrients to grow. We thus compared the internalization of AuNPs@mPEG in both breast cancer and normal mammary gland tissue cell lines. The 120 nm AuNPs@mPEG was used for the in vitro uptake owing to the reasons that we previously explained [13]. As expected, the quantity of uptake and subsequent distribution in tumor cells were different from those in the normal cells (Figure S5). Prominent black spots representing the aggregates of AuNPs, were present in cancer cells. Liu et al. reported that non-phagocytic HepG2 cells took up positively charged AuNPs, but not as many negatively-charged AuNPs, while phagocytic RAW264.7 cells efficiently took up both negatively- and positively-charged AuNPs [34]. Since the zeta-potential of 5 nm AuNPs@mPEG was − 14.9 mV (Table S1), there is no contradiction with this report.

Considering the short half-life of ²¹¹At, the difference in the uptake rate of AuNPs should significantly impact on the selective killing of tumors by ²¹¹At. The use of ²¹¹At-labeled AuNPs allows spatiotemporal selective uptake of radionuclides into tumor cells due to the high uptake capacity of the nanoparticles into tumor cells and passive targeting through the EPR effect.

We also investigated the gold nanoparticles (5 nm AuNPs@H16 and AuNPs@H16/RGD), that were modified with oligohistidine peptide [20] and cyclic RGD [21, 22], which can target tumor pH and the integrin αvβ3, respectively, to promote their tumor targeting ability and cellular uptake. RGD peptide, which can bind with the tumor marker integrin αvβ3, is widely used for targeted delivery [21, 22]. Oligohistidine peptides can be effectively internalized into cells, and can be used for tumor-targeted approaches due to their pH-responsive [20]. In vitro study revealed showed significant internalization and higher DSB induction of the two kinds peptides modified AuNPs.

However, the accumulation of both AuNPs@H16 and AuNPs@H16/RGD in tumors was not high but significantly higher in the liver compared with mPEG-modified AuNPs. Due to the high accumulation in the liver, these AuNPs are not applicable for treatments via intravenous administration. Considering the high internalization and significant DSB induction ability of the peptide-modified AuNPs, these are expected to be beneficial for treatments via local administration. In fact, Dziawer et al. reported that trastuzumab-modified ²¹¹At-labeled AuNPs, which exhibited high affinity and cytotoxicity to the HER2-overexpressing human ovarian SKOV-3 cell line [26], is a potential prospective tool for local treatment of HER2-positive breast cancer.

From a synthetic point of view, the simple operation and short-time synthesis of ²¹¹At-AuNPs are major advantages in the synthetic process. The ²¹¹At-labeled AuNPs can be easily obtained by simply mixing AuNPs and ²¹¹At solution, although the ²¹¹At solution was found to be a mixture of chemical species probably with different valences, such as At⁺, At⁻, etc. Various surface-modified ²¹¹At-labeled AuNPs were also readily obtained at RCY. In addition, the At-Au bond was reported to be stable both in theory and experimentally [13, 18, 34]. Another major problem was that ²¹¹At or ²¹¹At-labeled compounds readily stick to the reaction vessel and reduce the yields of labeled products. Nevertheless, the ²¹¹At adsorption capacity to AuNPs is stronger than that to the reaction vessel, and the adsorption amount of ²¹¹At in reaction tubes was quite low. In addition, because of high RCY, no purification was necessary which is useful when using short-lived isotopes.

Taking the above advantages into consideration, ²¹¹At-AuNPs seems to be very suitable for cancer treatment development and has potential clinical application in the future.

This study sheds new light on the substantial potential of AuNPs as carrier for the targeted delivery of ²¹¹At via intravenous administration. Our results showed that AuNP is a satisfactory carrier for ²¹¹At delivery, due to the simple and efficient synthesis process and high stability. Intravenous administration of 5 nm ²¹¹At-AuNPs@mPEG has a significant anti-tumor effect. We took advantage of the EPR effect and the fact that cancer cells have a higher nanoparticle uptake rate than normal cells, and successfully developed a novel TAT compound.

TAT: Targeted alpha-particle therapy, AuNPs: Gold nanoparticles, mPEG: Methoxy poly(ethylene glycol), H16 peptide: 2-Mercaptopropionic acid-HHHHH HHHHH HHHHH H-NH2 trifluoroacetate salt, RGD peptide: (cyclo[Arg-Gly-Asp-D-Phe-Lys(Cys)]), LET: Linear energy transfer, PB: Phosphate buffer, EPR: Enhanced permeability and retention, %ID: Percentage injected dose, %ID/g: Percentage of injected dose per gram, RCY: Radiochemical yield, DMEM: Dulbecco’s modified Eagle medium, PBS: Phosphate-buffered saline, UV-Vis: Ultraviolet-visible, DLS: Dynamic light scattering, TEM: Transmission electron microscopy, DSB: Double-strand break, PSMA: Prostate specific membrane antigen, FAP: fibroblast activation protein, FAPI: fibroblast activation protein inhibitor

Acknowledgements

In this study, we gratefully acknowledge Dr. Akihiro Ito at Analytical Instrument Facility, Graduate School of Science, Osaka University for support of TEM measurement. We would like to acknowledge the support from the multidisciplinary PhD program for pioneering quantum beam application and Osaka University Fellowship “Quantum Leader Recourses”. ²¹¹At was supplied through the Supply Platform of Short-lived Radioisotopes, supported by the JSPS Grant-in-Aid for Scientific Research on Innovative Areas (16H06278).

Authors’ contributions

X.H., K.K-N., Y.K., K.K., A.S. conceived the idea, planned the experiments and designed the research. X.H., K.K-N., Y.K. performed the experiments and prepared the figures. K.O., A.T., A.S., H.H., Y.W. produced ²¹¹At. K.F., K.K., A.S., H.K. supervised the study. X.H. wrote the main manuscript text and all authors discussed the results and contributed to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by JSPS KAKENHI, Grant Number, JP20H05675, JP20H00404 and 22J10766 (Grant-in-Aid for JSPS Fellows), QiSS program of the OPERA grant number JPMJOP1721 and The Uehara Memorial Foundation. ²¹¹At was supplied from RIKEN through the material transfer agreement and was given through Supply Platform of Short-lived Radioisotopes supported by JSPS Grant-in-Aid for Scientific Research on Innovative Areas, Grant Number JP16H06278.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the ARRIVE (Animal Research: Reporting In Vivo Experiments) and the Osaka University Animal Experiment Regulations, and approved by the Osaka University Animal Experiment Committee.

Consent for publication

All authors agree to be published.

Competing interests

The authors have no potential competing of interest to declare with respect to the conduct of the research, the authorship, and the publication of this article.

Author details

1. Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, 1-1 Machikaneyama, Osaka 560-0043, Japan

2. Division of Science, Institute for Radiation Sciences, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan

3. Core for Medicine and Science Collaborative Research and Education, Forefront Research Center, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

4. Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

5. Faculty of Health Science, Osaka Aoyama University, 2-11-1 Niina, Minoh, Osaka 562-8580, Japan

6. Nishina Center for Accelerator-Based Science, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

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No competing interests reported.

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Astatine-211-Labeled Gold Nanoparticles for Targeted Alpha-particle Therapy via Intravenous Injection

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Abstract

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Background

Methods

Astatine-211 production

AuNPs modification and characterization

In vivo biodistribution and therapy efficacy

Internalization evaluation

Statistical analysis

Results

Biodistribution study

In vivo therapeutic effect

Cellular uptake of AuNPs and DNA DSBs in vitro

Discussion

Conclusions

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References

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