Combining BNCT with carbonic anhydrase inhibition for mesothelioma treatment: Synthesis, in vitro, in vivo studies of ureidosulfamido carboranes.

Mesothelioma is a malignant neoplasm of mesothelial cells caused by exposure to asbestos. The average survival time after diagnosis is usually nine/twelve months. A multi-therapeutic approach is therefore required to treat and prevent recurrence. Boronated derivatives containing a carborane cage, a sulfamido group and an ureido functionality (CA-USF) have been designed, synthesised and tested, in order to couple Boron Neutron Capture Therapy (BNCT) and the inhibition of Carbonic Anhydrases (CAs), which are overexpressed in many tumours. In vitro studies showed greater inhibition than the reference drug acetazolamide (AZ). To increase solubility in aqueous media, CA-USFs were used as inclusion complexes of hydroxypropyl ß-cyclodextrin (HP-ß-CD) in all the inhibition and cell experiments. BNCT experiments carried out on AB22 (murine mesothelioma) cell lines showed a marked inhibition of cell proliferation by CA-USFs, and in one case a complete inhibition of proliferation twenty days after neutron irradiation. Finally, in vivo neutron irradiation experiments on a mouse model of mesothelioma demonstrated the efficiency of combining CA IX inhibition and BNCT treatment. Indeed, a greater reduction in tumour mass was observed in treated mice compared to untreated mice, with a significant higher effect when combined with BNCT. For in vivo experiments CA-USFs were administered as inclusion complexes of higher molecular weight ß-CD polymers thus increasing the selective extravasation into tumour tissue and reducing clearance. In this way, boron uptake was maximised and CA-USFs demonstrated to be in vivo well tolerated at a therapeutic dose. The therapeutic strategy herein described could be expanded to other cancers with increased CA IX activity, such as melanoma, glioma, and breast cancer.

A compact scintillator-based detector with collimator and shielding for dose monitoring in boron neutron capture therapy.

Boron neutron capture therapy exploits 10B(n,α)7Li reactions for targeted tumor destruction. In this work, we aimed at developing a dose monitoring system based on the detection of 478 keV gamma rays emitted by the reactions, which is very challenging due to the severe background present. We investigated a compact gamma-ray detector with a pinhole collimator and shielding housing. Experimental nuclear reactor measurements involved varying boron concentrations and artificial shifts of the sources. The system successfully resolved the 478 keV photopeak and detected 1 cm lateral displacements, confirming its suitability for precise boron dose monitoring.

Study of Alternative Imaging Methods for In Vivo Boron Neutron Capture Therapy.

Boron Neutron Capture Therapy (BNCT) is an innovative and highly selective treatment against cancer. Nowadays, in vivo boron dosimetry is an important method to carry out such therapy in clinical environments. In this work, different imaging methods were tested for dosimetry and tumor monitoring in BNCT based on a Compton camera detector. A dedicated dataset was generated through Monte Carlo tools to study the imaging capabilities. We first applied the Maximum Likelihood Expectation Maximization (MLEM) iterative method to study dosimetry tomography. As well, two methods based on morphological filtering and deep learning techniques with Convolutional Neural Networks (CNN), respectively, were studied for tumor monitoring. Furthermore, clinical aspects such as the dependence on the boron concentration ratio in image reconstruction and the stretching effect along the detector position axis were analyzed. A simulated spherical gamma source was studied in several conditions (different detector distances and boron concentration ratios) using MLEM. This approach proved the possibility of monitoring the boron dose. Tumor monitoring using the CNN method shows promising results that could be enhanced by increasing the training dataset.

A novel pH sensitive theranostic PLGA nanoparticle for boron neutron capture therapy in mesothelioma treatment.

This study aims to develop poly lactic-co-glycolic acid (PLGA) nanoparticles with an innovative imaging-guided approach based on Boron Neutron Capture Therapy for the treatment of mesothelioma. The herein-reported results demonstrate that PLGA nanoparticles incorporating oligo-histidine chains and the dual Gd/B theranostic agent AT101 can successfully be exploited to deliver a therapeutic dose of boron to mesothelioma cells, significantly higher than in healthy mesothelial cells as assessed by ICP-MS and MRI. The selective release is pH responsive taking advantage of the slightly acidic pH of the tumour extracellular environment and triggered by the protonation of imidazole groups of histidine. After irradiation with thermal neutrons, tumoral and healthy cells survival and clonogenic ability were evaluated. Obtained results appear very promising, providing patients affected by this rare disease with an improved therapeutic option, exploiting PLGA nanoparticles.

Potentialities of High-Resolution 3-D CZT Drift Strip Detectors for Prompt Gamma-Ray Measurements in BNCT.

Recently, new high-resolution cadmium-zinc-telluride (CZT) drift strip detectors for room temperature gamma-ray spectroscopic imaging were developed by our group. The CZT detectors equipped with orthogonal anode/cathode collecting strips, drift strips and dedicated pulse processing allow a detection area of 6 × 20 mm2 and excellent room temperature spectroscopic performance (0.82% FWHM at 661.7 keV). In this work, we investigated the potentialities of these detectors for prompt gamma-ray spectroscopy (PGS) in boron neutron capture therapy (BNCT). The detectors, exploiting the measurement of the 478 keV prompt gamma rays emitted by 94% 7Li nuclides from the 10B(n, α)7Li reaction, are very appealing for the development of single-photon emission computed tomography (SPECT) systems and Compton cameras in BNCT. High-resolution gamma-ray spectra from 10B samples under thermal neutrons were measured at the T.R.I.G.A. Mark II research nuclear reactor of the University of Pavia (Italy).

Cobaltabis(dicarbollide) ([o-COSAN]-) as Multifunctional Chemotherapeutics: A Prospective Application in Boron Neutron Capture Therapy (BNCT) for Glioblastoma.

PURPOSE: The aim of our study was to assess if the sodium salt of cobaltabis(dicarbollide) and its di-iodinated derivative (Na[o-COSAN] and Na[8,8'-I2-o-COSAN]) could be promising agents for dual anti-cancer treatment (chemotherapy + BNCT) for GBM. METHODS: The biological activities of the small molecules were evaluated in vitro with glioblastoma cells lines U87 and T98G in 2D and 3D cell models and in vivo in the small model animal Caenorhabditis elegans (C. elegans) at the L4-stage and using the eggs. RESULTS: Our studies indicated that only spheroids from the U87 cell line have impaired growth after treatment with both compounds, suggesting an increased resistance from T98G spheroids, contrary to what was observed in the monolayer culture, which highlights the need to employ 3D models for future GBM studies. In vitro tests in U87 and T98G cells conclude that the amount of 10B inside the cells is enough for BNCT irradiation. BNCT becomes more effective on T98G after their incubation with Na[8,8'-I2-o-COSAN], whereas no apparent cell-killing effect was observed for untreated cells. CONCLUSIONS: These small molecules, particularly [8,8'-I2-o-COSAN]-, are serious candidates for BNCT now that the facilities of accelerator-based neutron sources are more accessible, providing an alternative treatment for resistant glioblastoma.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

A Novel Approach to Design and Evaluate BNCT Neutron Beams Combining Physical, Radiobiological, and Dosimetric Figures of Merit.

(1) Background:The quality of neutron beams for Boron Neutron Capture Therapy (BNCT) is currently defined by its physical characteristics in air. Recommendations exist to define whether a designed beam is useful for clinical treatment. This work presents a new way to evaluate neutron beams based on their clinical performance and on their safety, employing radiobiological quantities. (2) Methods: The case study is a neutron beam for deep-seated tumors from a 5 MeV proton beam coupled to a beryllium target. Physical Figures of Merit were used to design five beams; however, they did not allow a clear ranking of their quality in terms of therapeutic potential. The latter was then evaluated based on in-phantom dose distributions and on the calculation of the Uncomplicated Tumor Control Probability (UTCP). The safety of the beams was also evaluated calculating the in-patient out-of-beam dosimetry. (3) Results: All the beams ensured a UTCP comparable to the one of a clinical beam in phantom; the safety criterion allowed to choose the best candidate. When this was tested in the treatment planning of a real patient treated in Finland, the UTCP was still comparable to the one of the clinical beam. (4) Conclusions: Even when standard physical recommendations are not met, radiobiological and dosimetric criteria demonstrate to be a valid tool to select an effective and safe beam for patient treatment.

Colocalization of tracks from boron neutron capture reactions and images of isolated cells.

In Boron Neutron Capture Therapy, the boronated drug plays a leading role in delivering a lethal dose to the tumour. The effectiveness depends on the boron macroscopic concentration and on its distribution at sub-cellular level. This work shows a way to colocalize alpha particles and lithium ions tracks with cells. A neutron autoradiography technique is used, which combines images of cells with images of tracks produced in a solid-state nuclear track detector.

In vitro and in vivo BNCT investigations using a carborane containing sulfonamide targeting CAIX epitopes on malignant pleural mesothelioma and breast cancer cells.

This study aims at merging the therapeutic effects associated to the inhibition of Carbonic Anhydrase IX (CAIX), an essential enzyme overexpressed by cancer cells including mesothelioma and breast cancer, with those ones brought by the application of Boron Neutron Capture Therapy (BNCT). This task was pursued by designing a sulfonamido-functionalised-carborane (CA-SF) that acts simultaneously as CAIX inhibitor and boron delivery agent. The CAIX expression, measured by Western blot analysis, resulted high in both mesothelioma and breast tumours. This finding was exploited for the delivery of a therapeutic dose of boron (> 20 µg/g) to the cancer cells. The synergic cytotoxic effects operated by the enzymatic inhibition and neutron irradiation was evaluated in vitro on ZL34, AB22 and MCF7 cancer cells. Next, an in vivo model was prepared by subcutaneous injection of AB22 cells in Balb/c mice and CA-SF was administered as inclusion complex with a ß-cyclodextrin oligomer. After irradiation with thermal neutrons tumour growth was evaluated for 25 days by MRI. The obtained results appear very promising as the tumour growth was definitively markedly lower in comparison to controls and the CAIX inhibitor alone. This approach appears promising and it call consideration for the design of new therapeutic routes to cure patients affected by this disease.

Recent advances in the development of high-resolution 3D cadmium-zinc-telluride drift strip detectors.

In the last two decades, great efforts have been made in the development of 3D cadmium-zinc-telluride (CZT) detectors operating at room temperature for gamma-ray spectroscopic imaging. This work presents the spectroscopic performance of new high-resolution CZT drift strip detectors, recently developed at IMEM-CNR of Parma (Italy) in collaboration with due2lab (Italy). The detectors (19.4 mm × 19.4 mm × 6 mm) are organized into collecting anode strips (pitch of 1.6 mm) and drift strips (pitch of 0.4 mm) which are negatively biased to optimize electron charge collection. The cathode is divided into strips orthogonal to the anode strips with a pitch of 2 mm. Dedicated pulse processing analysis was performed on a wide range of collected and induced charge pulse shapes using custom 32-channel digital readout electronics. Excellent room-temperature energy resolution (1.3% FWHM at 662 keV) was achieved using the detectors without any spectral corrections. Further improvements (0.8% FWHM at 662 keV) were also obtained through a novel correction technique based on the analysis of collected-induced charge pulses from anode and drift strips. These activities are in the framework of two Italian research projects on the development of spectroscopic gamma-ray imagers (10-1000 keV) for astrophysical and medical applications.

Design of a BNCT irradiation room based on proton accelerator and beryllium target.

Preliminary studies for the design of an accelerator-based BNCT clinical facility are presented. The Beam Shaping Assembly neutron activation was evaluated experimentally and with Monte Carlo simulations. The activations of patient, air and walls in the room, the absorbed doses by the patient and the in-air dose distributions were evaluated. Based on these calculations, different walls compositions were tested to optimize the environmental conditions. Borated concrete, advantageously reducing the thermal flux in the room, was proven the best choice.

Design, synthesis and preliminary in-vitro studies of novel boronated monocarbonyl analogues of Curcumin (BMAC) for antitumor and ß-amiloyd disaggregation activity.

Curcumin is currently being investigated for its capacity to treat many types of cancer and to prevent the neuron damage that is observed in Alzheimer's disease (AD). However, its clinical use is limited by its low stability and solubility in aqueous solutions. In this study, we propose a completely new class of boronated monocarbonyl analogues of Curcumin (BMAC, 6a-c), in which a carbonyl group replaces the Curcumin ß-diketone functionality, and an ortho-carborane, an icosahedral boron cluster, substitutes one of the two phenolic rings. BMAC antitumor activity against MCF7 and OVCAR-3 cell lines was assessed in vitro and compared to that of Curcumin and the corresponding MAC derivative. BMAC 6a-c showed efficiencies that are comparable to that of MAC and superior to that of Curcumin in both the cell lines. Moreover, the inhibition of the formation of ß-amyloid aggregates by BMAC 6a-c was evaluated and it was shown that compound 6c, which contains two OH moieties, has a better efficiency than Curcumin. The presence of a second -OH group can enhance the compound's binding efficacy with ß-amyloid aggregates. For the future, the presence of at least one carborane group means that the BMAC antitumor effect can be coupled with Boron Neutron Capture Therapy.

Combining magnetic nanoparticles and icosahedral boron clusters in biocompatible inorganic nanohybrids for cancer therapy.

The potential biomedical applications of the MNPs nanohybrids coated with m-carboranylphosphinate (1-MNPs) as a theranostic biomaterial for cancer therapy were tested. The cellular uptake and toxicity profile of 1-MNPs from culture media by human brain endothelial cells (hCMEC/D3) and glioblastoma multiform A172 cell line were demonstrated. Prior to testing 1-MNPs' in vitro toxicity, studies of colloidal stability of the 1-MNPs' suspension in different culture media and temperatures were carried out. TEM images and chemical titration confirmed that 1-MNPs penetrate into cells. Additionally, to explore 1-MNPs' potential use in Boron Neutron Capture Therapy (BNCT) for treating cancer locally, the presence of the m-carboranyl coordinated with the MNPs core after uptake was proven by XPS and EELS. Importantly, thermal neutrons irradiation in BNCT reduced by 2.5 the number of cultured glioblastoma cells after 1-MNP treatment, and the systemic administration of 1-MNPs in mice was well tolerated with no major signs of toxicity.

Charged particle spectrometry to measure 10B concentration in bone.

Osteosarcoma is the most common primary malignant tumour of bone in young patients. The survival of these patients has largely been improved due to adjuvant and neo-adjuvant chemotherapy in addition to surgery. Boron neutron capture therapy (BNCT) is proposed as a complementary therapy, due to its ability to inactivate tumour cells that may survive the standard treatment and that may be responsible for recurrences and/or metastases. BNCT is based on neutron irradiation of a tumour enriched in 10B with a boron-loaded drug. Low-energy neutron capture in 10B creates charged particles that impart a high dose to tumour cells, which can be calculated only knowing the boron concentration. Charged particle spectrometry is a method that can be used to quantify boron concentration. This method requires acquisition of the energy spectra of charged particles such as alpha particles produced by neutron capture reactions in thin tissue sections irradiated with low-energy neutrons. Boron concentration is then determined knowing the stopping power of the alpha particles in the sample material. This paper describes the adaptation of this method for bone, with emphasis on sample preparation, experimental set-up and stopping power assessment of the involved alpha particles. The knowledge of boron concentration in healthy bones is important, because it allows for any dose limitation that might be necessary to avoid adverse effects such as bone fragility. The measurement process was studied through Monte Carlo simulations and analytical calculations. Finally, the boron content of bone samples was measured by alpha spectrometry at the TRIGA reactor in Pavia, Italy, and compared to that obtained by neutron autoradiography. The agreement between the results obtained with these techniques confirms the suitability of alpha spectrometry to measure boron in bone.

An innovative therapeutic approach for malignant mesothelioma treatment based on the use of Gd/boron multimodal probes for MRI guided BNCT.

The aim of this study is to develop an innovative imaging guided approach based on Boron Neutron Capture Therapy, for the treatment of mesothelioma, assisted by the quantification of the in vivo boron distribution by MRI. The herein reported results demonstrate that overexpressed Low Density Lipoproteins receptors can be successfully exploited to deliver to mesothelioma cells a therapeutic dose of boron (26 µg/g), significantly higher than in the surrounding tissue (3.5 µg/g). Boron and Gd cells uptake was assessed by ICP-MS and MRI on two mesothelioma (ZL34, AE17) and two healthy (MRC-5 and NMuMg) cell lines. An in vivo model was prepared by subcutaneous injection of ZL34 cells in Nu/Nu mice. After irradiation with thermal neutrons, tumor growth was evaluated for 40 days by MRI. Tumor masses of boron treated mice showed a drastic reduction of about 80-85%. The obtained results appear very promising providing patients affected by this rare disease with an improved therapeutic option, exploiting LDL transporters.

Understanding the potentiality of accelerator based-boron neutron capture therapy for osteosarcoma: dosimetry assessment based on the reported clinical experience.

BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumour, and its incidence is higher in children and adolescents, for whom it represents more than 10% of solid cancers. Despite the introduction of adjuvant and neo-adjuvant chemotherapy that markedly increased the success rate in the treatment, aggressive surgery is still needed and a considerable percentage of patients do not survive due to recurrences or early metastases. Boron Neutron Capture Therapy (BNCT), an experimental radiotherapy, was investigated as a treatment that could allow a less aggressive surgery by killing infiltrated tumour cells in the surrounding healthy tissues. BNCT requires an intense neutron beam to ensure irradiation times of the order of 1 h. In Italy, a Radio Frequency Quadrupole (RFQ) proton accelerator has been designed and constructed for BNCT, and a suitable neutron spectrum was tailored by means of Monte Carlo calculations. This paper explores the feasibility of BNCT to treat osteosarcoma using this neutron source based on accelerator. METHODS: The therapeutic efficacy of BNCT was analysed evaluating the dose distribution obtained in a clinical case of femur osteosarcoma. Mixed field dosimetry was assessed with two different formalisms whose parameters were specifically derived from radiobiological experiments involving in vitro UMR-106 osteosarcoma cell survival assays and boron concentration assessments in an animal model of osteosarcoma. A clinical case of skull osteosarcoma treated with BNCT in Japan was re-evaluated from the point of view of dose calculation and used as a reference for comparison. RESULTS: The results in the case of femur osteosarcoma show that the RFQ beam would ensure a suitable tumour dose painting in a total irradiation time of less than an hour. Comparing the dosimetry between the analysed case and the treated patient in Japan it turns out that doses obtained in the femur tumour are at least as good as the ones delivered in the skull osteosarcoma. The same is concluded when the comparison is carried out taking into account osteosarcoma irradiations with photon radiation therapy. CONCLUSIONS: The possibility to apply BNCT to osteosarcoma would allow a multimodal treatment consisting in neo-adjuvant chemotherapy, high-LET selective radiation treatment and a more conservative surgery.

Theranostic Nanoparticles Loaded with Imaging Probes and Rubrocurcumin for Combined Cancer Therapy by Folate Receptor Targeting.

The combination of different therapeutic modalities is a promising option to combat the recurrence of tumors. In this study, polylactic and polyglycolic acid nanoparticles were used for the simultaneous delivery of a boron-curcumin complex (RbCur) and an amphiphilic gadolinium complex into tumor cells with the aim of performing boron and gadolinium neutron capture therapy (NCT) in conjunction with the additional antiproliferative effects of curcumin. Furthermore, the use of Gd complexes allows magnetic resonance imaging (MRI) assessment of the amount of B and Gd internalized by tumor cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were targeted to ovarian cancer (IGROV-1) cells through folate receptors, by including in the formulation a PEGylated phospholipid functionalized with the folate moiety. NCT was performed on IGROV-1 cells internalizing 6.4 and 78.6 µg g-1 of 10 B and 157 Gd, respectively. The synergic action of neutron treatment and curcumin cytotoxicity was shown to result in a significant therapeutic improvement.

Insights into the use of gadolinium and gadolinium/boron-based agents in imaging-guided neutron capture therapy applications.

Gadolinium neutron capture therapy (Gd-NCT) is currently under development as an alternative approach for cancer therapy. All of the clinical experience to date with NCT is done with (10)B, known as boron neutron capture therapy (BNCT), a binary treatment combining neutron irradiation with the delivery of boron-containing compounds to tumors. Currently, the use of Gd for NCT has been getting more attention because of its highest neutron cross-section. Although Gd-NCT was first proposed many years ago, its development has suffered due to lack of appropriate tumor-selective Gd agents. This review aims to highlight the recent advances for the design, synthesis and biological testing of new Gd- and B-Gd-containing compounds with the task of finding the best systems able to improve the NCT clinical outcome.

An improved neutron autoradiography set-up for (10)B concentration measurements in biological samples.

AIM: Boron Neutron Capture Therapy (BNCT) is a binary hadrontherapy which exploits the neutron capture reaction in boron, together with a selective uptake of boronated substances by the neoplastic tissue. There is increasing evidence that future improvements in clinical BNCT will be triggered by the discovery of new boronated compounds, with higher selectivity for the tumor with respect to clinically used sodium borocaptate (BSH) and boronophenylalanine (BPA). BACKGROUND: Therefore, a (10)B quantification technique for biological samples is needed in order to evaluate the performance of new boronated formulations. MATERIALS AND METHODS: This article describes an improved neutron autoradiography set-up employing radiation sensitive films where the latent tracks are made visible by proper etching conditions. RESULTS: Calibration curves for both liquid and tissue samples were obtained. CONCLUSIONS: The obtained calibration curves were adopted to set-up a mechanism to point out boron concentration in the whole sample.