Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part II. Toxicity, Pharmacokinetics and Biodistribution

Abstract

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of ²²³Ra-labeled and PSMA-targeted NaA nanozeolites [²²³RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of ¹³³Ba- and ²²³Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to ²²³Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of ²²³Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.

Keywords: D2B antibodies; PSMA-targeted radioligand therapy; biodistribution; pharmacokinetics; prostate cancer; radium-223; toxicity; zeolite nanoparticles.

Figure A1

Elimination of radioactivity from blood of BALB/c mice injected intravenously (tail vein) with 133BaA-silane-PEG (black line) and 133BaA-silane-PEG-anty-PSMA (red line).

Figure A2

Comparative analysis of biodistribution of ²²³Ra-silane-PEG-D2B and ²²³Ra-silane-PEG with biodistribution of ²²³RaCl₂ (Xofigo) on 7th day. BALB/c mice (weight: 19.1–23.6 g; age 6 week) were exposed to a single intravenous dose of ²²³RaCl2 of ~25 μL (20 g body weight with solution activity at 500 kBq/mL on day 0).

Figure 1

Scheme of ²²³Ra-labeled nanozeolite particle functionalization with silane-PEG group and anti-PSMA D2B antibody.

Figure 2

The effect of NaA-silane-PEG and NaA-silane-PEG-D2B on the proportion of viable cells (annexin V-negative and propidium iodide-negative cells), early apoptotic cells (annexin V-positive cells and propidium iodide-negative cells) and late apoptotic/necrotic cells (annexin V-positive and propidium iodide-positive cells). LNCaP C4-2 cells (A), DU-145 cells (B), RWPE-1 cells (C) and HPrEC cells (D) were exposed to 50 mcg mL⁻¹ of NaA-silane-PEG or NaA-silane-PEG-D2B for 96 h. Data are expressed as the mean ± standard deviation (SD) from three independent experiments. * p < 0.05 versus control group.

Figure 3

The effect of NaA-silane-PEG and NaA-silane-PEG-D2B on the proportion of caspase 3/7 positive cells. LNCaP C4-2 cells, DU-145 cells, RWPE-1 cells and HPrEC cells were exposed to 50 mcg mL⁻¹ of NaA-silane-PEG or NaA-silane-PEG-D2B for 96 h. Data are expressed as the mean ± standard deviation (SD) from three independent experiments. * p < 0.05 versus control group.

Figure 4

Venn diagrams depicting number of genes significantly affected (p < 0.05) by NaA-silanePEG and NaA-silane-PEG-D2B nanocarriers in LNCaP C4-2, DU-145, RWPE-1 and HPrEC cells. The effect of NaA-silane-PEG on number of genes involved in autoimmune and inflammatory responses (A) and in NF-κB signaling (B). The effect of NaA-silane-PEG-D2B on the number of genes involved in autoimmune and inflammatory responses (C) and in NF-κB signaling (D).

Figure 5

Statistically significant changes in NF-κB signaling and in the inflammatory response and autoimmunity gene expression with a cut-off value for gene expression fold changes >1.5 in DU-145 cells, RWPE-1 and HPrEC cells after treatment with 50 mcg mL⁻¹ of NaA-silane-PEG for 96 h. Mean fold change values from three independent experiments are presented. Error bars represent a 95% confidence interval.

Figure 6

Statistically significant changes in NF-κB signaling and in the inflammatory response and autoimmunity gene expression with a cut-off value for gene expression fold changes >1.5 in (A) LNCaP C4-2, DU-145 cells, RWPE-1 and (B) HPrEC cells after treatment with 50 mcg mL⁻¹ of NaA-silane-PEG-D2B for 96 h. Mean fold change values from three independent experiments are presented. Error bars represent a 95% confidence interval.

Figure 7

Influence of unlabeled and radiolabeled compounds on hematological and biochemical parameters of blood and body weight 7 days after a single injection of the tested compounds in BALB/c mice. Body weight (a), concentration of ALT and AST enzymes (b), concentration of platelets (c), hematological parameters in blood of mice exposed to NaA-silane-PEG (d) and NaA-silane-PEG-D2B (e). Data are expressed as the mean ± standard deviation (SD) from 10 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control group.

Figure 8

Representative images of bone marrow, kidneys and liver sections after exposure of BALB/c mice to NaA-silane-PEG-D2B and ²²³RaA-silane-PEG-D2B for 7 days. Bone marrow (A), kidneys (B) and liver (C) sections from control (PBS) mice. Bone marrow (D), kidneys (E) and liver (F) sections from mice after exposure to NaA-silane-PEG-D2B. Bone marrow (G), kidneys (H) and liver (I) sections from mice after exposure to ²²³RaA-silane-PEG-D2B. Scale bars, 200 mcm.