Enhancing Boron Neutron Capture Therapy (BNCT) with Materials Based on COSAN-Functionalized Nanoparticles

Abstract

Background/Objectives: Boron neutron capture therapy (BNCT) is a promising approach for selectively targeting and destroying malignant cells using ¹⁰B isotopes. A significant challenge in BNCT lies in the development of efficient boron delivery systems that ensure adequate boron accumulation within tumor cells. This study aims to synthesize, characterize, and evaluate COSAN-functionalized nanoparticles (NP@I-COSAN) as a potential boron carrier for BNCT. Methods: Hybrid nanoparticles were synthesized by conjugating monoiodinated cobaltabisdicarbollides (I-COSAN) to commercially available acrylic polymer-based nanoparticles. Functionalization and cellular uptake were confirmed through FTIR, TGA, UV-Vis spectroscopy, and TEM/EDX analyses. Biocompatibility was evaluated by assessing cytotoxicity in HeLa cells and C. elegans as an in vivo model. Intracellular boron uptake was quantified using ICP-MS, with results compared to those obtained with 4-borono-L-phenylalanine conjugated to fructose (f-BPA). An in vitro BNCT proof-of-concept assay was also performed to evaluate therapeutic efficacy. Results: NP@I-COSAN demonstrated low cytotoxicity and efficient internalization in cells. ICP-MS analysis revealed stable boron retention, comparable to traditional boron agents. The BNCT assay further showed that NP@I-COSAN was effective in inducing tumor cell apoptosis, even at lower boron concentrations than conventional treatments. Conclusions: These results underscore the potential of NP@I-COSAN as an effective boron delivery system for BNCT, offering a promising strategy to enhance boron accumulation within tumor cells and improve treatment efficacy.

Keywords: BNCT; C. elegans; boron clusters; metallacarboranes.

Scheme 1

Representative scheme on the functionalization of NPs with I-COSAN, leading to the formation of NP@I-COSAN.

Figure 1

(A) Bar graphs indicating the percentage of HeLa cell survival after 24 h (left) and 48 h (right) of treatment with four different concentrations (10, 25, 50, and 100 μg∙mL⁻¹) of NP@I-COSAN and the positive control (DMSO 10 vol. %), error bars mean ± 5%. (B) Fluorescence microscopy photographs of the LIVE/DEAD experiments of HeLa cells after being incubated for 24 h (left) and 48 h (right) with 100 μg∙mL⁻¹ NP@I-COSAN.

Figure 2

TEM images of the accumulation of NP@I-COSAN inside HeLa cells after 48 h of incubation with solutions of 0.05 M of NP@I-COSAN.

Figure 3

In vivo evaluation of I-COSAN and NP@I-COSAN in the C. elegans model. (A) Optical microscope images of control and I-COSAN- and NP@I-COSAN-treated worms. The right pictures are insets with a higher magnification than the left image. The worm’s head and tail are highlighted in the left inset. Arrows indicate the presence of NP@I-COSAN. The scale bar on the right is 100 µm, and the scale bar on the left is 50 µm. (B) Biodistribution of NP@I-COSAN. Percentage of worms with NP@I-COSAN in each of the four parts (pharynx, anterior gut, central gut, and posterior gut). Each region was represented with a gradient color from 0 to 100%. (C) Survival rate after 24 h of incubation with S-basal buffer (control) and 25 µg/mL I-COSAN and NP@I-COSAN (N = 3, n = 150). (D) The length of the worms was evaluated after 24 h of nanomaterial exposure (N = 3, n = 50). * p < 0.05, ** p < 0.01.

Figure 4

Boron uptake in HeLa cells was evaluated after exposure to 0.045 ppm of ¹⁰B-NP@I-COSAN and 25 ppm of ¹⁰BPA-f for 2 and 4 h. (**) p < 0.01 and (***) p < 0.001, according to the two-way ANOVA analysis (Tukey’s test).

Figure 5

(A) Cell survival after treatment with 0.045 ppm of ¹⁰B-NP@I-COSAN and 25 ppm of ¹⁰BPA-f, post-neutron irradiation (2 Gy). (B) Dosimetry for thermal neutron irradiation without and with boron (25 ppm ¹⁰B). Note: Dosimetry for the BNCT groups is based on BPA-f, which has a boron-10 concentration of 25 ppm. (*) p < 0.05, according to the two-way ANOVA analysis (Tukey’s test).

Figure 6

(A) Quantitative analysis of fluorescence microscopy at 48 h post-irradiation; (B) Post-irradiation analyses of apoptotic and necrotic events (dead cells) were performed using fluorescence microscopy. Representative images of stained cells were taken 48 h after irradiation. Apoptotic nuclei, indicated by Hoechst staining (red arrows), exhibited features such as peripheral chromatin condensation, cytoplasmic blebbing, and fragmentation. The cytoplasm of viable cells was labeled with 4,5-diaminofluorescein (DAF), whereas necrotic cells were identified using propidium iodide (IP) staining (red arrows). (*) p < 0.05, (**) p < 0,01, (***) p < 0.001, according to the two-way ANOVA analysis (Tukey’s test).