The Rise of Boron-Containing Compounds: Advancements in Synthesis, Medicinal Chemistry, and Emerging Pharmacology.

Boron-containing compounds (BCC) have emerged as important pharmacophores. To date, five BCC drugs (including boronic acids and boroles) have been approved by the FDA for the treatment of cancer, infections, and atopic dermatitis, while some natural BCC are included in dietary supplements. Boron's Lewis acidity facilitates a mechanism of action via formation of reversible covalent bonds within the active site of target proteins. Boron has also been employed in the development of fluorophores, such as BODIPY for imaging, and in carboranes that are potential neutron capture therapy agents as well as novel agents in diagnostics and therapy. The utility of natural and synthetic BCC has become multifaceted, and the breadth of their applications continues to expand. This review covers the many uses and targets of boron in medicinal chemistry.

A bis-boron boramino acid PET tracer for brain tumor diagnosis.

PURPOSE: Boramino acids are a class of amino acid biomimics that replace the carboxylate group with trifluoroborate and can achieve the 18F-labeled positron emission tomography (PET) and boron neutron capture therapy (BNCT) with identical chemical structure. METHODS: This study reports a trifluoroborate-derived boronophenylalanine (BBPA), a derived boronophenylalanine (BPA) for BNCT, as a promising PET tracer for tumor imaging. RESULTS: Competition inhibition assays in cancer cells suggested the cell accumulation of [18F]BBPA is through large neutral amino acid transporter type-1 (LAT-1). Of note, [18F]BBPA is a pan-cancer probe that shows notable tumor uptake in B16-F10 tumor-bearing mice. In the patients with gliomas and metastatic brain tumors, [18F]BBPA-PET shows good tumor uptake and notable tumor-to-normal brain ratio (T/N ratio, 18.7 ± 5.5, n = 11), higher than common amino acid PET tracers. The [18F]BBPA-PET quantitative parameters exhibited no difference in diverse contrast-enhanced status (P = 0.115-0.687) suggesting the [18F]BBPA uptake was independent from MRI contrast-enhancement. CONCLUSION: This study outlines a clinical trial with [18F]BBPA to achieve higher tumor-specific accumulation for PET, provides a potential technique for brain tumor diagnosis, and might facilitate the BNCT of brain tumors.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

PURPOSE: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9 µm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. METHOD: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. RESULT: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. CONCLUSION: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Assessing the effectiveness of fluorinated and α-methylated 3-boronophenylalanine for improved tumor-specific boron delivery in boron neutron capture therapy.

A [10B]boron agent and a nuclear imaging probe for pharmacokinetic estimation form the fundamental pair in successful boron neutron capture therapy (BNCT). However, 4-[10B]borono-l-phenylalanine (BPA), used in clinical BNCT, has undesirable water solubility and tumor selectivity. Therefore, we synthesized fluorinated and α-methylated 3-borono-l-phenylalanine (3BPA) derivatives to realize improved water solubility, tumor targetability, and biodistribution. All 3BPA derivatives exhibited over 10 times higher water solubility than BPA. Treatment with α-methylated 3BPA derivatives resulted in decreased cell uptake via l-type amino acid transporter (LAT) 2 while maintaining LAT1 recognition, thereby significantly improving LAT1/LAT2 selectivity. Biodistribution studies showed that fluorinated α-methyl 3BPA derivatives exhibited reduced boron accumulation in nontarget tissues, including muscle, skin, and plasma. Consequently, these derivatives demonstrated significantly improved tumor-to-normal tissue ratios compared to 3BPA and BPA. Overall, fluorinated α-methyl 3BPA derivatives with the corresponding radiofluorinated compounds hold potential as promising agents for future BNCT/PET theranostics.

Macrophages as carriers of boron carbide nanoparticles dedicated to boron neutron capture therapy.

BACKGROUND: The use of cells as carriers for the delivery of nanoparticles is a promising approach in anticancer therapy, mainly due to their natural properties, such as biocompatibility and non-immunogenicity. Cellular carriers prevent the rapid degradation of nanoparticles, improve their distribution, reduce cytotoxicity and ensure selective delivery to the tumor microenvironment. Therefore, we propose the use of phagocytic cells as boron carbide nanoparticle carriers for boron delivery to the tumor microenvironment in boron neutron capture therapy. RESULTS: Macrophages originating from cell lines and bone marrow showed a greater ability to interact with boron carbide (B4C) than dendritic cells, especially the preparation containing larger nanoparticles (B4C 2). Consequently, B4C 2 caused greater toxicity and induced the secretion of pro-inflammatory cytokines by these cells. However, migration assays demonstrated that macrophages loaded with B4C 1 migrated more efficiently than with B4C 2. Therefore, smaller nanoparticles (B4C 1) with lower toxicity but similar ability to activate macrophages proved to be more attractive. CONCLUSIONS: Macrophages could be promising cellular carriers for boron carbide nanoparticle delivery, especially B4C 1 to the tumor microenvironment and thus prospective use in boron neutron capture therapy.

Lithium salts cytotoxicity and accumulation in melanoma cells in vitro.

Boron neutron capture therapy is a perspective selective technology for the destruction of cancer cells, while the use of lithium instead of boron may represent a new and promising vector for the development of neutron capture therapy (NCT). The aim of the study was a comparative assessment of the cytotoxicity of various lithium salts, as well as an analysis of the accumulation of lithium in tumor cells in vitro to determine the possibility of using lithium in NCT. The cytotoxicity of lithium salts was determined using MTT-test and colony forming assay on human fibroblasts BJ-5ta, human skin melanoma SK-Mel-28, and mouse skin melanoma B16 cell lines. An assessment of lithium concentration in cells was performed using inductively coupled plasma atomic emission spectrometry. Our results showed that three different lithium salts at a concentration of 40 µg/ml are not toxic for both tumor and normal cells. The highest uptake values were obtained on murine melanoma B16 cells when exposed to lithium carbonate (0.8 µg/106 cells); however, human melanoma SK-Mel-28 cells effectively accumulated both lithium carbonate and lithium citrate (about 0.46 µg/106 cells for two salts). Thus, our results demonstrate a range of non-toxic doses of lithium salts and a high uptake of lithium by tumor cells, which indicates the possibility to use the lithium in NCT.

Next generation of boron neutron capture therapy (BNCT) agents for cancer treatment.

Boron neutron capture therapy (BNCT) is one of the most promising treatments among neutron capture therapies due to its long-term clinical application and unequivocally obtained success during clinical trials. Boron drug and neutron play an equivalent crucial role in BNCT. Nevertheless, current clinically used l-boronophenylalanine (BPA) and sodium borocaptate (BSH) suffer from large uptake dose and low blood to tumor selectivity, and that initiated overwhelm screening of next generation of BNCT agents. Various boron agents, such as small molecules and macro/nano-vehicles, have been explored with better success. In this featured article, different types of agents are rationally analyzed and compared, and the feasible targets are shared to present a perspective view for the future of BNCT in cancer treatment. This review aims at summarizing the current knowledge of a variety of boron compounds, reported recently, for the application of BCNT.

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy.

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.

Measurement of an evaporation coefficient in tissue sections as a correction factor for 10B determination.

Boron neutron capture therapy (BNCT) is a cancer treatment option that combines preferential uptake of a boron compound in tumors and irradiation with thermal neutrons. For treatment planning, the boron concentration in different tissues must be considered. Neutron autoradiography using nuclear track detectors (NTD) can be applied to study both the concentration and microdistribution of boron in tissue samples. Histological sections are obtained from frozen tissue by cryosectioning. When the samples reach room temperature, they undergo an evaporation process, which leads to an increase in the boron concentration. To take this effect into account, certain correction factors (evaporation coefficients, CEv) must be applied. With this aim, a protocol was established to register and analyze mass variation of tissue sections, measured with a semimicro scale. Values of ambient temperature, pressure, and humidity were simultaneously recorded. Reproducible results of evaporation curves and CEv values were obtained for different tissue samples, which allowed the systematization of the procedure. This study could contribute to a more precise determination of boron concentration in tissue samples through the neutron autoradiography technique, which is of great relevance to make dosimetric calculations in BNCT.

HER-2-Targeted Boron Neutron Capture Therapy with Carborane-integrated Immunoliposomes Prepared via an Exchanging Reaction.

Boron neutron capture therapy (BNCT) is a promising modality for cancer treatment because of its minimal invasiveness. To maximize the therapeutic benefits of BNCT, the development of efficient platforms for the delivery of boron agents is indispensable. Here, carborane-integrated immunoliposomes were prepared via an exchanging reaction to achieve HER-2-targeted BNCT. The conjugation of an anti-HER-2 antibody to carborane-integrated liposomes successfully endowed these liposomes with targeting properties toward HER-2-overexpressing human ovarian cancer cells (SK-OV3); the resulting BNCT activity toward SK-OV3 cells obtained using the current immunoliposomal system was 14-fold that of the l-BPA/fructose complex, which is a clinically available boron agent. Moreover, the growth of spheroids treated with this system followed by thermal neutron irradiation was significantly suppressed compared with treatment with the l-BPA/fructose complex.

Rational Design, Multistep Synthesis and in Vitro Evaluation of Poly(glycerol) Functionalized Nanodiamond Conjugated with Boron-10 Cluster and Active Targeting Moiety for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT), advanced cancer treatment utilizing nuclear fission of 10 B atom in cancer cells, is attracting increasing attention. As 10 B delivery agent, sodium borocaptate (10 BSH, 10 B12 H11 SH ⋅ 2Na), has been used in clinical studies along with L-boronophenylalanine. Recently, this boron cluster has been conjugated with lipids, polymers or nanoparticles to increase selectivity to and retentivity in tumor. In this work, anticancer nanoformulations for BNCT are designed, consisting of poly(glycerol) functionalized detonation nanodiamonds (DND-PG) as a hydrophilic nanocarrier, the boron cluster moiety (10 B12 H11 2- ) as a dense boron-10 source, and phenylboronic acid or RGD peptide as an active targeting moiety. Some hydroxy groups in PG were oxidized to carboxy groups (DND-PG-COOH) to conjugate the active targeting moiety. Some hydroxy groups in DND-PG-COOH were then transformed to azide to conjugate 10 B12 H11 2- through click chemistry. The nanodrugs were evaluated in vitro using B16 murine melanoma cells in terms of cell viability, BNCT efficacy and cellular uptake. As a result, the 10 B12 H11 2- moiety is found to facilitate cellular uptake probably due to its negative charge. Upon thermal neutron irradiation, the nanodrugs with 10 B12 H11 2- moiety exhibited good anticancer efficacies with slight differences with and without targeting moiety.

Sweet Battle of the Epimers─Continued Exploration of Monosaccharide-Derived Delivery Agents for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a cancer therapy in which boron delivery agents play a crucial role. In theory, delivery agents with high tumor targeting capabilities can lead to selective eradication of tumor cells without causing harmful side effects. We have been working on a GLUT1-targeting strategy to BNCT for a number of years and found multiple promising hit compounds which outperform the clinically employed boron delivery agents in vitro. Herein, we continue our work in the field by further diversification of the carbohydrate scaffold in order to map the optimal stereochemistry of the carbohydrate core. In the sweet battle of the epimers, carborane-bearing d-galactose, d-mannose, and d-allose are synthesized and subjected to in vitro profiling studies─with earlier work on d-glucose serving as the reference. We find that all of the monosaccharide delivery agents display a significantly improved boron delivery capacity over the delivery agents approved for clinical use in vitro, thus providing a sound foundation for advancing toward in vivo preclinical assessment studies.

Evaluation of a Tumor-Targeting Oligosaccharide Nanosystem in BNCT on an Orthotopic Hepatocellular Carcinoma Model.

Boron neutron capture therapy (BNCT) is becoming a promising radiation treatment technique dealing with tumors due to its cellular targeting specificity. In this article, based on the biocompatible chitosan oligosaccharide (COS), we designed a boron delivery system using carborane (CB) as a boron drug with cRGD peptide modification and paclitaxel (PTX) loaded in the hydrophobic core. The nanoparticles (cRGD-COS-CB/PTX) realized the boron delivery into tumor sites with an enhanced permeability and retention (EPR) effect and an active targeting effect achieved by the cRGD-integrin interaction on the surface of tumor cells. The uniform spherical nanoparticles can be selectively taken by hepatoma cells rather than normal hepatocytes. In vivo experiments showed that the nanoparticles had a targeting effect on tumor sites in both subcutaneous and orthotopic tumor models, which was an encouraging result for radiotherapy for liver cancer. To sum up, the nanoparticles we produced proved to be promising dual-functionalized nanoparticles for radiotherapy and chemotherapy.

Development of a Gadolinium-Boron-Conjugated Albumin for MRI-Guided Neutron Capture Therapy.

Noninvasive monitoring of boron agent biodistribution is required in advance of neutron capture therapy. In this study, we developed a gadolinium-boron-conjugated albumin (Gd-MID-BSA) for MRI-guided neutron capture therapy. Gd-MID-BSA was prepared by labeling bovine serum albumin with a maleimide-functionalized gadolinium complex and a maleimide-functionalized closo-dodecaborate orthogonally. The accumulation of Gd-MID-BSA in tumors in CT26 tumor-bearing mice reached a maximum at 24 h after the injection, as confirmed by T1-based MRI and biodistribution analysis using inductively coupled plasma optical emission spectrometry. The concentrations of boron and gadolinium in the tumors exceeded the thresholds required for boron neutron capture therapy (BNCT) and gadolinium neutron capture therapy (GdNCT), respectively. The boron concentration ratios of tumor to blood and tumor to normal tissues satisfied the clinical criteria, indicating the reduction of undesired nuclear reactions of endogenous nuclei. The molar ratio of boron to gadolinium in the tumor was close to that of Gd-MID-BSA, demonstrating that the accumulation of Gd-MID-BSA in the tumor can be evaluated by MRI. Thermal neutron irradiation with Gd-MID-BSA resulted in significant suppression of tumor growth compared to the group injected with a boron-conjugated albumin without gadolinium (MID-BSA). The neutron irradiation with Gd-MID-BSA did not cause apparent side effects. These results demonstrate that the conjugation of gadolinium and boron within the albumin molecule offers a novel strategy for enhancing the therapeutic effect of BNCT and the potential of MRI-guided neutron capture therapy as a promising treatment for malignant tumors.

A Potential Boron Neutron Capture Therapy Agent Selectively Suppresses High-Grade Glioma: In Vitro and in Vivo Exploration.

Glioblastoma (GBM), as the most central nervous system (CNS) intractable disease, has spoiled millions of lives due to its high mortality. Even though several efforts have been made, the existing treatments have had limited success. In this sense, we studied a lead compound, the boron-rich selective epidermal growth factor receptor (EGFR)-inhibitor hybrid 1, as a potential drug for GBM treatment. For this end, we analyzed the in vitro activity of hybrid 1 in a glioma/primary astrocytes coculture, studying cellular death types triggered by treatment with this compound and its cellular localizations. Additionally, hybrid 1 concentrated boron in glioma cells selectively and more effectively than the boron neutron capture therapy (BNCT)-clinical agent 10B-l-boronophenylalanine and thus displayed a better in vitro-BNCT effect. This encouraged us to analyze hybrid 1 in vivo. Therefore, immunosuppressed mice bearing U87 MG human GBM were treated with both 1 and 1 encapsulated in a modified liposome (recognized by brain-blood barrier peptide transporters), and we observed a potent in vivo per se antitumor activity (tumor size decrease and animal survival increase). These data demonstrate that 1 could be a promising new targeted therapy for GBM.

Evaluating the biological effectiveness of boron neutron capture therapy by using microfluidics-based pancreatic tumor spheroids.

Background: The recent success of boron neutron capture therapy (BNCT) for cancer treatment has attracted considerable attention. Because irradiated neutrons penetrate deep into solid tumor tissue, BNCT efficacy is strongly influenced by cell pathophysiology in tumors. The tumor microenvironment critically influences tumor pathophysiology, but its effects on BNCT remain unexplored. Methods: We used a pancreatic tumor as a model to develop a high-throughput 3D tumor spheroid platform for evaluating BNCT efficacy under different microenvironment conditions. We expanded our system to serve as a transwell-like device in order to investigate the influence of stromal fibroblasts in the tumor microenvironment. Results: With the use of the proposed microfluidic chip and a laboratory pipette, more than 40 spheroids with controllable diameters (standard deviation <10%) could be cultured on a chip for more than 10 days. The response to BNCT from each spheroid can be monitored in real time. By using pancreatic tumor spheroids of two different diameters, we found that large spheroids, characterized by more hypoxic microenvironments, exhibited lower BNCT susceptibility. The cells in the hypoxic region expressed the HIF1-α signal, which is crucial in many therapeutic resistance signal pathways. In addition, the heterogeneous presence of stemness markers (Oct-4, Sox-2, and CD 44) implied that the underlying BNCT resistance mechanism was sophisticated. In the presence of fibroblasts, we found an association between ß-catenin nuclear translocation and BNCT resistance; membrane contacts from fibroblasts were found to be indispensable for translocation activation. Conclusions: In summary, by means of easily accessible microfluidic engineering, we developed tumor spheroids to recapture the pathophysiological characteristics of pancreatic tumors. Our data suggest that hypoxia and fibrosis can reduce BNCT efficacy in pancreatic cancer treatment. Considering the growing requirement for drug screening in personalized medicine, our findings and the developed method are expected to improve the fundamental understanding of BNCT and facilitate broad applications of BNCT in clinical settings.

Salvage boron neutron capture therapy for pediatric patients with recurrent diffuse midline glioma.

PURPOSE: Pediatric diffuse malignant glioma located in the brainstem was officially named "diffuse midline glioma" (DMG) by the World Health Organization in 2016. For this disease, radical surgery is not beneficial, and the only major treatment strategy is radiotherapy. However, the dose limitations to brainstem tissue mean that treatment by radiotherapy can only control and not eradicate the tumors, and there is no effective treatment for recurrence, resulting in short overall survival of 6-12 months. This paper reports our experience with boron neutron capture therapy (BNCT), a new treatment process, and its efficacy in treating children with recurrent DMG. METHODS: From September 2019 to July 2022, we treated 6 children affected by recurrent DMG. With the collaboration of Taipei Veteran General Hospital (TVGH) and National Tsing-Hua University (NTHU), each patient received two sessions of BNCT within 1 month. RESULTS: Among the six patients, three showed partial response and the rest had stable disease after the treatment. The overall survival and recurrence-free survival duration after treatment were 6.39 and 4.35 months, respectively. None of the patients developed severe side effects, and only one patient developed brain necrosis, which was most likely resulted from previous hypofractionated radiotherapy received. CONCLUSION: BNCT elicited sufficient tumor response with low normal tissue toxicity; it may benefit vulnerable pediatric patients with DMG.

Carborane bearing pullulan nanogel-boron oxide nanoparticle hybrid for boron neutron capture therapy.

Boron neutron capture therapy shows is a promising approach to cancer therapy, but the delivery of effective boron agents is challenging. To address the requirements for efficient boron delivery, we used a hybrid nanoparticle comprising a carborane = bearing pullulan nanogel and hydrophobized boron oxide nanoparticle (HBNGs) enabling the preparation of highly concentrated boron agents for efficient delivery. The HBNGs showed better anti-cancer effects on Colon26 cells than a clinically boron agent, L-BPA/fructose complex, by enhancing the accumulation and retention amount of the boron agent within cells in vitro. The accumulation of HBNGs in tumors, due to the enhanced permeation and retention effect, enabled the delivery of boron agents with high tumor selectivity, meeting clinical demands. Intravenous injection of boron neutron capture therapy (BNCT) using HBNGs decreased tumor volume without significant body weight loss, and no regrowth of tumor was observed three months after complete regression. The therapeutic efficacy of HBNGs was better than that of L-BPA/fructose complex. BNCT with HBNGs is a promising approach to cancer therapeutics.

Nanocomposite Materials Based on Polylactide and Gold Complex Compounds for Absorbed Dose Diagnostics in BNCT.

In this study, approaches to the synthesis of complex compound of gold with cysteine [AuCys]n for measuring absorbed dose in boron neutron capture therapy (BNCT) were developed. The dependence of the complex particle size on pH were established. Nanocomposite materials based on polylactide containing [AuCys]n particles with an average size of about 20 nm were obtained using the crazing mechanism. The structure of obtained materials was studied by electron microscopy. The release kinetics of [AuCys]n from polymer matrix were investigated. Release of [AuCys]n from the volume of the polymeric matrix had a delayed start-this process began only after 24 h and was characterized by an effective rate constant of 1 µg/h from a 20 mg composite sample. At the same time, in vitro studies showed that the concentration of 6.25 µg/mL was reliably safe and did not reduce the survival of U251 and SW-620 cells.