Nicoletta Protti

MRI-guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low-density lipoprotein transporters.

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group.

Design, development and characterization of multi-functionalized gold nanoparticles for biodetection and targeted boron delivery in BNCT applications

The aim of this study is to optimize targeted boron delivery to cancer cells and its tracking down to the cellular level. To this end, we describe the design and synthesis of novel nanovectors that double as targeted boron delivery agents and fluorescent imaging probes. Gold nanoparticles were coated with multilayers of polyelectrolytes functionalized with the fluorescent dye (FITC), boronophenylalanine and folic acid. In vitro confocal fluorescence microscopy demonstrated significant uptake of the nanoparticles in cancer cells that are known to overexpress folate receptors.

Synthesis of a carborane-containing cholesterol derivative and evaluation as a potential dual agent for MRI/BNCT applications

In this study the synthesis and characterization of a new dual, imaging and therapeutic, agent is proposed with the aim of improving the efficacy of Boron Neutron Capture Therapy (BNCT) in cancer treatment. The agent (Gd-B-AC01) consists of a carborane unit (ten boron atoms) bearing a cholesterol unit on one side (to pursue the incorporation into the liposome bi-layer) and a Gd(iii)/1,4,7,10-tetraazacyclododecane monoamide complex on the other side (as a MRI reporter to attain the quantification of the B/Gd concentration). In order to endow the BNCT agent with specific delivery properties, the liposome embedded with the MRI/BNCT dual probes has been functionalized with a pegylated phospholipid containing a folic acid residue at the end of the PEG chain. The vector allows the binding of the liposome to folate receptors that are overexpressed in many tumor types, and in particular, in human ovarian cancer cells (IGROV-1). An in vitro test on IGROV-1 cells demonstrated that Gd-B-AC01 loaded liposomes are efficient carriers for the delivery of the MRI/BNCT probes to the tumor cells. Finally, the BNCT treatment of IGROV-1 cells showed that the number of surviving cells was markedly smaller when the cells were irradiated after internalization of the folate-targeted GdB10-AC01/liposomes.

A theranostic approach based on the use of a dual boron/Gd agent to improve the efficacy of Boron Neutron Capture Therapy in the lung cancer treatment

This study aims at developing an innovative theranostic approach for lung tumor and metastases treatment, based on Boron Neutron Capture Therapy (BNCT). It relies on to the use of low density lipoproteins (LDL) as carriers able to maximize the selective uptake of boron atoms in tumor cells and, at the same time, to quantify the in vivo boron distribution by magnetic resonance imaging (MRI). Tumor cells uptake was initially assessed by ICP-MS and MRI on four types of tumor (TUBO, B16-F10, MCF-7, A549) and one healthy (N-MUG) cell lines. Lung metastases were generated by intravenous injection of a Her2+ breast cancer cell line (i.e. TUBO) in BALB/c mice and transgenic EML4-ALK mice were used as primary tumor model. After neutron irradiation, tumor growth was followed for 30-40 days by MRI. Tumor masses of boron treated mice increased markedly slowly than the control group. From the clinical editor: In this article, the authors described an improvement to existing boron neutron capture therapy. The dual MRI/BNCT agent, carried by LDLs, was able to maximize the selective uptake of boron in tumor cells, and, at the same time, quantify boron distribution in tumor and in other tissues using MRI. Subsequent in vitro and in vivo experiments showed tumor cell killing after neutron irradiation.

Boron uptake measurements in a rat model for Boron Neutron Capture Therapy of lung tumours.

Lung carcinoma is the leading cause of cancer mortality in the Western countries. Despite the introduction over the last few years of new therapeutic agents, survival from lung cancer has shown no discernible improvement in the last 20 years. For these reasons any efforts to find and validate new effective therapeutic procedures for lung cancer are very timely. The selective boron uptake in the tumour with respect to healthy tissues makes Boron Neutron Capture Therapy a potentially advantageous option in the treatment of tumours that affect whole vital organs, and that are surgically inoperable. To study the possibility of applying BNCT to the treatment of diffuse pulmonary tumours, an animal model for boron uptake measurements in lung metastases was developed. Both healthy and tumour-bearing rats were infused with Boronophenylalanine (BPA) and sacrificed at different time intervals after drug administration. The lungs were extracted, and prepared for boron analysis by neutron autoradiography and α-spectroscopy. The boron concentrations in tumour and normal lung were plotted as a function of the time elapsed after BPA administration. The concentration in tumour is almost constant within the error bars for all the time intervals of the experiment (1-8 h), while the curve in normal lung decreases after 4 h from BPA infusion. At 4 h, the ratio of boron concentration in tumour to boron concentration in healthy lung is higher than 3, and it stays above this level up to 8 h. Also the images of boron distribution in the samples, obtained by neutron autoradiography, show a selective absorption in the metastases.

Calculations of dose distributions in the lungs of a rat model irradiated in the thermal column of the TRIGA reactor in Pavia

To test the possibility to apply boron neutron capture therapy (BNCT) to lung tumors, some rats are planned to be irradiated in the thermal column of the TRIGA reactor of the University of Pavia. Before the irradiation, lung metastases will be induced in BDIX rats, which will be subsequently infused with boronophenylalanine (BPA). During the irradiation, the rats will be positioned in a box designed to shield the whole animal except the thorax area. In order to optimize the irradiation set-up and to design a suitable shielding box, a set of calculations were performed with the MCNP Monte Carlo transport code. A rat model was constructed using the MCNP geometry capabilities and was positioned in a box with walls filled with lithium carbonate. A window was opened in front of the lung region. Different shapes of the holder and of the window were tested and analyzed in terms of the dose distribution obtained in the lungs and of the dose absorbed by the radiosensitive organs in the rat. The best configuration of the holder ensures an almost uniform thermal neutron flux inside the lungs (Phi(max)/Phi(min)=1.5), an irradiation time about 10 min long, to deliver at least 40 Gy(w) to the tumor, a mean lung dose of 5.9+/-0.4 Gy(w), and doses absorbed by all the other healthy tissues below the tolerance limits.

A Model of Radiation-Induced Cell Killing: Insights into Mechanisms and Applications for Hadron Therapy

A mechanism-based, two-parameter biophysical model of cell killing was developed with the aim of elucidating the mechanisms underlying radiation-induced cell death and predicting cell killing by different radiation types, including protons and carbon ions at energies and doses of interest for cancer therapy. The model assumed that certain chromosome aberrations (dicentrics, rings and large deletions, called “lethal aberrations”) lead to clonogenic inactivation, and that aberrations derive from μm-scale misrejoining of chromatin fragments, which in turn are produced by “dirty” double-strand breaks called “cluster lesions” (CLs). The average numbers of CLs per Gy per cell were left as a semi-free parameter and the threshold distance for chromatin-fragment rejoining was defined the second parameter. The model was “translated” into Monte Carlo code and provided simulated survival curves, which were compared with survival data on V79 cells exposed to protons, carbon ions and X rays. The agreement was good between simulations and survival data and supported the assumptions of the model at least for doses up to a few Gy. Dicentrics, rings and large deletions were found to be lethal not only for AG1522 cells exposed to X rays, as already reported by others, but also for V79 cells exposed to protons and carbon ions of different energies. Furthermore, the derived CL yields suggest that the critical DNA lesions leading to clonogenic inactivation are more complex than “clean” DSBs. After initial validation, the model was applied to characterize the particle and LET dependence of proton and carbon cell killing. Consistent with the proton data, the predicted fraction of inactivated cells after 2 Gy protons was 40–50% below 7.7 keV/μm, increased by a factor ∼1.6 between 7.7–30.5 keV/μm, and decreased by a factor ∼1.1 between 30.5–34.6 keV/μm. These LET values correspond to proton energies below a few MeV, which are always present in the distal region of hadron therapy spread-out Bragg peaks (SOBP). Consistent with the carbon data, the predicted fraction of inactivated cells after 2 Gy carbon was 40–50% between 13.7–32.4 keV/μm, it increased by a factor ∼1.7 between 32.4–153.5 keV/μm, and decreased by a factor ∼1.1 between 153.5–339.1 keV/μm. Finally, we applied the model to predict cell death at different depths along a carbon SOBP used for preclinical experiments at HIMAC in Chiba, Japan. The predicted fraction of inactivated cells was found to be roughly constant (less than 10%) along the SOBP, suggesting that this approach may be applied to predict cell killing of therapeutic carbon beams and that, more generally, dicentrics, rings and deletions at the first mitosis may be regarded as a biological dose for these beams. This study advanced our understanding of the mechanisms of radiation-induced cell death and characterized the particle and LET dependence of proton and carbon cell killing along a carbon SOBP. The model does not use RBE values, which can be a source of uncertainty. More generally, this model is a mechanism-based tool that in minutes can predict cell inactivation by protons or carbon ions of a given energy and dose, based on an experimental photon curve and in principle, a single (experimental) survival point for the considered ion type and energy.

Testing and linearity calibration of films of phenol compounds exposed to thermal neutron field for EPR dosimetry.

This paper reports the preliminary results obtained by Electron Paramagnetic Resonance (EPR) measurements on films of IRGANOX® 1076 phenols with and without low content (5% by weight) of gadolinium oxide (Gd2O3) exposed in the thermal column of the Triga Mark II reactor of LENA (Laboratorio Energia Nucleare Applicata) of Pavia (Italy). Thanks to their size, the phenolic films here presented are good devices for the dosimetry of beams with high dose gradient and which require accurate knowledge of the precise dose delivered. The dependence of EPR signal as function of neutron dose was investigated in the fluence range between 10(11) cm(-2) and 10(14) cm(-2). Linearity of EPR response was found and the signal was compared with that of commercial alanine films. Our analysis showed that gadolinium oxide (5% by weight) can enhance the thermal neutron sensitivity more than 18 times. Irradiated dosimetric films of phenolic compound exhibited EPR signal fading of about 4% after 10 days from irradiation.

In Vitro and In Vivo Studies of Boron Neutron Capture Therapy: Boron Uptake/Washout and Cell Death

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal‐neutron irradiation of cells enriched with 10B, which produces &agr; particles and 7Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron‐enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron‐loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation‐induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.

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 10B and 157Gd, respectively. The synergic action of neutron treatment and curcumin cytotoxicity was shown to result in a significant therapeutic improvement.