15N Hyperpolarization of Metronidazole Antibiotic in Aqueous Media Using Phase-Separated Signal Amplification by Reversible Exchange with Parahydrogen.

Metronidazole is a prospective hyperpolarized MRI contrast agent with potential hypoxia sensing utility for applications in cancer, stroke, neurodegenerative diseases, etc. We demonstrate a pilot procedure for production of ∼30 mM hyperpolarized [15N3]metronidazole in aqueous media by using a phase-separated SABRE-SHEATH hyperpolarization method, with nitrogen-15 polarization exceeding 2.2% on all three 15N sites achieved in less than 2 min. The 15N polarization T1 of ∼12 min is reported for the 15NO2 group at the clinically relevant field of 1.4 T in the aqueous phase, demonstrating a remarkably long lifetime of the hyperpolarized state. The produced aqueous solution of [15N3]metronidazole that contained only ∼100 µM of residual Ir was deemed biocompatible via validation through the MTT colorimetric test for assessing cell metabolic activity using human embryotic kidney HEK293T cells. This low-cost and ultrafast hyperpolarization procedure represents a major advance for the production of a biocompatible HP [15N3]metronidazole (and potentially other hyperpolarized drugs) formulation for MRI sensing applications.

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.

Laser-Synthesized Elemental Boron Nanoparticles for Efficient Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is one of the most appealing radiotherapy modalities, whose localization can be further improved by the employment of boron-containing nanoformulations, but the fabrication of biologically friendly, water-dispersible nanoparticles (NPs) with high boron content and favorable physicochemical characteristics still presents a great challenge. Here, we explore the use of elemental boron (B) NPs (BNPs) fabricated using the methods of pulsed laser ablation in liquids as sensitizers of BNCT. Depending on the conditions of laser-ablative synthesis, the used NPs were amorphous (a-BNPs) or partially crystallized (pc-BNPs) with a mean size of 20 nm or 50 nm, respectively. Both types of BNPs were functionalized with polyethylene glycol polymer to improve colloidal stability and biocompatibility. The NPs did not initiate any toxicity effects up to concentrations of 500 µg/mL, based on the results of MTT and clonogenic assay tests. The cells with BNPs incubated at a 10B concentration of 40 µg/mL were then irradiated with a thermal neutron beam for 30 min. We found that the presence of BNPs led to a radical enhancement in cancer cell death, namely a drop in colony forming capacity of SW-620 cells down to 12.6% and 1.6% for a-BNPs and pc-BNPs, respectively, while the relevant colony-forming capacity for U87 cells dropped down to 17%. The effect of cell irradiation by neutron beam uniquely was negligible under these conditions. Finally, to estimate the dose and regimes of irradiation for future BNCT in vivo tests, we studied the biodistribution of boron under intratumoral administration of BNPs in immunodeficient SCID mice and recorded excellent retention of boron in tumors. The obtained data unambiguously evidenced the effect of a neutron therapy enhancement, which can be attributed to efficient BNP-mediated generation of α-particles.

Carborane-Containing Folic Acid bis-Amides: Synthesis and In Vitro Evaluation of Novel Promising Agents for Boron Delivery to Tumour Cells.

The design of highly selective low-toxic, low-molecular weight agents for boron delivery to tumour cells is of decisive importance for the development of boron neutron capture therapy (BNCT), a modern efficient combined method for cancer treatment. In this work, we developed a simple method for the preparation of new closo- and nido-carborane-containing folic acid bis-amides containing 18-20 boron atoms per molecule. Folic acid derivatives containing nido-carborane residues were characterised by high water solubility, low cytotoxicity, and demonstrated a good ability to deliver boron to tumour cells in in vitro experiments (up to 7.0 µg B/106 cells in the case of U87 MG human glioblastoma cells). The results obtained demonstrate the high potential of folic acid-nido-carborane conjugates as boron delivery agents to tumour cells for application in BNCT.

Dose-Dependent Suppression of Human Glioblastoma Xenograft Growth by Accelerator-Based Boron Neutron Capture Therapy with Simultaneous Use of Two Boron-Containing Compounds.

(1) Background: Developments in accelerator-based neutron sources moved boron neutron capture therapy (BNCT) to the next phase, where new neutron radiation parameters had to be studied for the treatment of cancers, including brain tumors. We aimed to further improve accelerator-BNCT efficacy by optimizing dosimetry control, beam parameters, and combinations of boronophenylalanine (BPA) and sodium borocaptate (BSH) administration in U87MG xenograft-bearing immunodeficient mice with two different tumor locations. (2) Methods: The study included two sets of experiments. In Experiment #1, BPA only and single or double irradiation in higher doses were used, while, in Experiment #2, BPA and BSH combinations and single or double irradiation with dosage adjustment were analyzed. Mice without treatment or irradiation after BPA or BPA+BSH injection were used as controls. (3) Results: Irradiation parameter adjustment and BPA and BSH combination led to 80-83% tumor-growth inhibition index scores, irradiation:BNCT ratios of 1:2, and increases in animal life expectancy from 9 to 107 days. (4) Conclusions: Adjustments in dosimetry control, calculation of irradiation doses, and combined use of two 10B compounds allowed for BNCT optimization that will be useful in the development of clinical-trial protocols for accelerator-based BNCT.

In Vitro and In Vivo Evaluation of Fluorescently Labeled Borocaptate-Containing Liposomes.

Boron neutron capture therapy (BNCT), a binary cancer therapeutic modality, has moved to a new phase since development of accelerator-based neutron sources and establishment of BNCT centers in Finland and Japan. That stimulated efforts for better boron delivery agent development. As liposomes have shown effective boron delivery properties and sufficient tumor retention, fluorescent liposome labelling may serve as a rapid method to study initial ability of newly synthesized liposomes to be captured by tumor cells prior to experiments on boron accumulation and neutron irradiation. In this work, we studied the accumulation and biodistribution of pegylated liposomes with encapsulated borocaptate (BSH) and a fluorescent label (Nile Red) in U87 (human glioblastoma), SW-620 (human colon carcinoma), SK-MEL-28 (human melanoma), FetMSC (mesenchymal human embryo stem cells), and EMBR (primary embryocytes) cell lines as well as an orthotopic xenograft model of U87 glioma in SCID mice. Results indicate that fluorescent microscopy is effective at determining the intracellular localization of the liposomes using a fluorescent label. The synthesized, pegylated liposomes showed higher accumulation in tumors compared to normal cells, with characteristic concentration peaks in SW-620 and U87 cell lines, and provided in vivo tumor selectivity with several-fold higher tumor tissue fluorescence at the 6-h timepoint. Graphical abstract Fluorescent images of U-87 glioma cells after 24 hours of incubation with BSH-containing liposomes labeled with lipophilic Nile Red (red color)and water-soluble FITC-Dextran (green color); cell nuclei in blue color (DAPI-staining) (×400). Scale bar is 50 µm. Fluorescent labelling serves as anexpress method to study liposome delivery efficiency prior to boron accumulation evaluation and BNCT irradiation experiments.

Accelerator-based boron neutron capture therapy for malignant glioma: a pilot neutron irradiation study using boron phenylalanine, sodium borocaptate and liposomal borocaptate with a heterotopic U87 glioblastoma model in SCID mice.

Purpose: To evaluate the efficacy of boron neutron capture therapy (BNCT) for a heterotopic U87 glioblastoma model in SCID mice using boron phenylalanine (BPA), sodium borocaptate (BSH) and liposomal BSH as boron compounds at a unique, accelerator-based neutron source.Materials and methods: Glioblastoma models were obtained by subcutaneous implantation of U87 cells in the right thighs of SCID mice before administration of 350 mg/kg of BPA (BPA-group), 100 mg/kg of BSH (BSH-group) or 100 mg/kg of BSH in PEGylated liposomes (liposomal BSH-group) into the retroorbital sinus. Liposomes were prepared by reverse-phase evaporation. Neutron irradiation was carried out at a proton accelerator with a lithium target developed for BNCT at the Budker Institute of Nuclear Physics, Novosibirsk, Russian Federation. A proton beam current integral of 3 mA/h and energy of 2.05 MeV were used for neutron generation.Results: Boron compound accumulation in tumor tissues at the beginning of irradiation was higher in the BPA group, followed by the Liposomal BSH and BSH groups. Tumor growth was significantly slower in all irradiated mice from the 7th day after BNCT compared to untreated controls (p < .05). Tumor growth in all treated groups showed no large variation, apart from the Irradiation only group and the BPA group on the 7th day after BNCT. The overall trend of tumor growth was clear and the differences between treatment groups became significant from the 50th day after BNCT. Tumor growth was significantly slower in the Liposomal BSH group compared to the Irradiation only group on the 50th (p = .012), 53rd (p = .005), and the 57th (p = .021) days after treatment. Tumor growth in the Liposomal BSH group was significantly different from that in the BPA group on the 53rd day after BNCT (p = .021) and in the BSH group on the 50th (p = .024), 53rd (p = .015), and 57th (p = .038) days after BNCT. Skin reactions in the form of erosions and ulcers in the tumor area developed in treated as well as untreated animals with further formation of fistulas and necrotic decay cavities in most irradiated mice.Conclusions: We observed a tendency of BNCT at the accelerator-based neutron source to reduce or suspend the growth of human glioblastoma in immunodeficient animals. Liposomal BSH showed better long-term results compared to BPA and non-liposomal BSH. Further modifications in liposomal boron delivery are being studied to improve treatment outcomes.

pH-triggered delivery of magnetic nanoparticles depends on tumor volume.

Nowadays there is growing recognition of the fact that biological systems have a greater impact on nanoparticle target delivery in tumors than nanoparticle design. Here we investigate the targeted delivery of Fe3O4 magnetic nanoparticles conjugated with pH-low-insertion peptide (MNP-pHLIP) on orthotopically induced MDA-MB-231 human breast carcinoma xenografts of varying volumes as a model of cancer progression. Using in vivo magnetic resonance imaging and subsequent determination of iron content in tumor samples by inductively coupled plasma atomic emission spectroscopy we found that MNP-pHLIP accumulation depends on tumor volume. Transmission electron microscopy, histological analysis and immunohistochemical staining of tumor samples suggest that blood vessel distribution is the key factor in determining the success of the accumulation of nanoparticles in tumors.

Oncolytic Effect of Adenoviruses Serotypes 5 and 6 Against U87 Glioblastoma Cancer Stem Cells.

BACKGROUND/AIM: Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS: Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS: Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. CONCLUSION: Ad6 was shown to be a promising agent for treating glioblastomas.

Design of protein homocystamides with enhanced tumor uptake properties for (19)F magnetic resonance imaging.

Straightforward and reliable tools for in vivo imaging of tumors can benefit the studies of cancer development, as well as contribute to successful diagnosis and treatment of cancer. (19)F NMR offers an exceptional quantitative way of in vivo imaging of the infused agents because of the lack of (19)F signals from the endogenous molecules in the body. The purpose of this study is to develop molecular probes with appropriate NMR characteristics and the biocompatibility for in vivo applications using (19)F MRI. We have studied the reaction between perfluorotoluene and homocysteine thiolactone resulting in the formation of N-substituted homocysteine thiolactone derivative. It has been shown that the reaction occurs selectively at the para position. This fluorine-labeled homocysteine thiolactone has been employed for the introduction of a perfluorotoluene group as a (19)F-containing tag into human serum albumin. The modified protein has been studied in terms of its ability to aggregate and promote the formation of free radicals. By comparing the properties of N-perfluorotoluene-homocystamide of albumin with N-homocysteinylated albumin, it has been revealed that blocking of the alpha-amino group of the homocysteine residue in the fluorinated albumin conjugate inhibits the dangerous aggregation process, as well as free radical formation. A dual-labeled albumin-based molecular probe for (19)F MRI and fluorescence microscopy has been obtained by functionalizing the protein with both maleimide of a fluorescent dye and a fluorinated thiolactone derivative. The incubation of cells with this conjugate did not reveal any significant reduction in cell viability with respect to the parent albumin. The perfluorotoluene-labeled albumin has been demonstrated to act as a promising agent for in vivo (19)F MRI.

Genetically delivered antibody protects against West Nile virus.

Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.

Neutralizing monoclonal antibodies against Russian strain of the West Nile virus.

We have developed a panel of 16 hybridomas secreting neutralizing monoclonal antibodies (Nt- MAbs) to Russian isolate (LEIV-Vlg99-27889-human) of the West Nile virus (WNV). Most of the Nt-Mabs were either IgG1 or IgG3 subtypes. Nine of the 16 neutralizing MAbs detected WNV protein E in Western blot. According to their Nt-activities, Western blot results and cross-reactivity, the MAbs were divided into four groups. Monoclonal antibodies from group I were able to neutralize WNV strains Vlg99-27889, Vlg00-27924, Hp-94, A-1640, A-72, Tur-2914, and Eg101. The Nt-activity of MAbs from groups II-IV towards these WNV strains was variable. Recombinant fragments E(1-180), E(1-321), and E(260-466) of protein E were used for preliminary mapping of domains recognized by Nt-MAbs. Only five Nt-MAbs were able to react with the recombinant polypeptides. The MAbs 9E2, 7G9, 11G3, and 7E6 from group Ia recognized Nt-epitope(s) between amino acids 321 and 466 of protein E and Nt-MAb 4F11 (group III) reacted with residues 1-180. This demonstrates that two discrete regions of protein E are involved in neutralization of WNV. Our data on immunochemical, biological activities of Nt-MAbs and mapping of Nt-epitopes using recombinant polypeptides suggest at least 13 different Nt-epitopes for WNV.