Organosilica nanoparticles containing sodium borocaptate (BSH) provide new prospects for boron neutron capture therapy (BNCT): efficient cellular uptake and enhanced BNCT efficacy.

Boron neutron capture therapy (BNCT), a method based on the fission of boron-10 upon neutron irradiation, has emerged as an attractive option for radiation therapy. To date, the main drugs used in BNCT are 4-boronophenylalanine (BPA) and sodium borocaptate (BSH). While BPA has been extensively tested in clinical trials, the use of BSH has been limited, mainly due to its poor cellular uptake. Here, we describe a novel type of mesoporous silica-based nanoparticle containing BSH covalently attached to a nanocarrier. Synthesis and characterization of these nanoparticles (BSH-BPMO) are presented. The synthetic strategy involves a click thiol-ene reaction with the boron cluster, providing hydrolytically stable linkage with the BSH in four steps. The BSH-BPMO nanoparticles were efficiently taken up into cancer cells and accumulated in the perinuclear region. Inductively coupled plasma (ICP) measurements of boron uptake in cells highlight the important role of the nanocarrier in the enhancement of boron internalization. BSH-BPMO nanoparticles were also taken up and distributed throughout tumour spheroids. BNCT efficacy was examined by the neutron exposure of the tumour spheroids. BSH-BPMO loaded spheroids were completely destroyed upon neutron irradiation. In contrast, neutron irradiation of tumour spheroids loaded with BSH or BPA resulted in significantly less spheroid shrinkage. The significant difference in BNCT efficacy of the BSH-BPMO was correlated with the improved boron uptake via the nanocarrier. Overall, these results demonstrate the critical role of the nanocarrier in BSH internalization and the enhanced BNCT efficacy of the BSH-BPMO compared with BSH and BPA, two drugs used in BNCT clinical trials.

Auger electrons and DNA double-strand breaks studied by using iodine-containing chemicals.

Irradiation of high Z elements such as iodine, gold, gadolinium with monochromatic X-rays causes photoelectric effects that include the release of Auger electrons. Decay of radioactive iodine such as I-123 and I-125 also results in multiple events and some involve the generation of Auger electrons. These electrons have low energy and travel only a short distance but have a strong effect on DNA damage including the generation of double-strand breaks. In this chapter, we focus on iodine and discuss various studies that used iodine-containing chemicals to generate Auger electrons and cause DNA double-strand breaks. First, DNA synthesis precursors containing iodine were used to place iodine on DNA. DNA binding dyes such as iodine Hoechst were investigated for Auger electron generation and DNA breaks. More recently, iodine containing nanoparticles were developed. We describe our study using tumor spheroids loaded with iodine nanoparticles and synchrotron-generated monochromatic X-rays. This study led to the demonstration that an optimum effect on DNA double-strand break formation is observed with a 33.2keV X-ray which is just above the K-edge energy of iodine.

Tumor Accumulation of PIP-Based KRAS Inhibitor KR12 Evaluated by the Use of a Simple, Versatile Chicken Egg Tumor Model.

BACKGROUND: The KRAS inhibitor KR12, based on pyrrole-imidazole polyamide (PIP), has been developed and shown to exhibit efficacy in mouse experiments. Because some PIP species exhibit tumor accumulation capability, we decided to evaluate whether the PIP portion of KR12 exhibits tumor accumulation. We employed the CAM assay that provides a simple method for tumor accumulation evaluation. METHODS: KR12 PIP was synthesized and conjugated to TAMRA to produce a fluorescently labeled reagent (KR12-TAMRA). This reagent was injected into a fertilized chicken egg that has been transplanted with human cancer cells. Distribution of the red fluorescence was examined by cutting out tumor as well as various organs from the embryo. RESULTS: The red fluorescence of KR12-TAMRA was found to overlap with the green fluorescence of the tumor formed with GFP-expressing cancer cells. We also observed nuclear localization of KR12-TAMRA. Treatment of KR12 that contained the alkylating agent CBI in the tumor-bearing chicken egg resulted in tumor growth inhibition. CONCLUSIONS: KR12 contains a PIP that has two key features: tumor accumulation and nuclear localization. KR12 conjugated with CBI exhibits inhibition of tumor growth in the CAM model.

Iodine containing porous organosilica nanoparticles trigger tumor spheroids destruction upon monochromatic X-ray irradiation: DNA breaks and K-edge energy X-ray.

X-ray irradiation of high Z elements causes photoelectric effects that include the release of Auger electrons that can induce localized DNA breaks. We have previously established a tumor spheroid-based assay that used gadolinium containing mesoporous silica nanoparticles and synchrotron-generated monochromatic X-rays. In this work, we focused on iodine and synthesized iodine-containing porous organosilica (IPO) nanoparticles. IPO were loaded onto tumor spheroids and the spheroids were irradiated with 33.2 keV monochromatic X-ray. After incubation in CO2 incubator, destruction of tumor spheroids was observed which was accompanied by apoptosis induction, as determined by the TUNEL assay. By employing the γH2AX assay, we detected double strand DNA cleavages immediately after the irradiation. These results suggest that IPO first generate double strand DNA breaks upon X-ray irradiation followed by apoptosis induction of cancer cells. Use of three different monochromatic X-rays having energy levels of 33.0, 33.2 and 33.4 keV as well as X-rays with 0.1 keV energy intervals showed that the optimum effect of all three events (spheroid destruction, apoptosis induction and generation of double strand DNA breaks) occurred with a 33.2 keV monochromatic X-ray. These results uncover the preferential effect of K-edge energy X-ray for tumor spheroid destruction mediated by iodine containing nanoparticles.

Various CAM tumor models.

Many types of in vivo animal tumor models have been established. Among these, the chicken chorioallantoic membrane (CAM) model has proven to be particularly useful for transplanting various types of cancer cell lines or tumor tissues to study tumor formation, angiogenesis and metastasis. The CAM model is useful as an animal tumor model, as tumor could be rapidly formed. The tumor formation occurs in 3-4 days, much faster than in mouse models. In addition, the CAM model can be used for drug screening for cancer therapy. This chapter provides an overview of various types of CAM tumor models.

An Xq22.1q22.2 nullisomy in a male patient with severe neurological impairment.

The proteolipid protein 1 gene (PLP1) is located on chromosome Xq22.2 and is related to X-linked recessive leukoencephalopathy (Pelizaeus-Merzbacher disease: PMD). Compared to PLP1 duplications, which are a major contributor to PMD, chromosomal deletions in this region are rare and only a few PMD patients with small deletions have been reported, suggesting that large deletions of this region would cause embryonic lethality. Previously, we have reported female patients, with chromosomal deletions in this region, who showed severe developmental delays and behavioral abnormalities. In this study, we identified the first case of a male patient associated with an Xq22 nullisomy in a region proximal to PLP1. The patient showed severe neurological impairment and was bedridden. Brain magnetic resonance imaging revealed a severely reduced cerebral volume. The chromosomal region proximal to PLP1 was considered to be significantly important for brain development.

Preferential expression of a bromoperoxidase in sporophytes of a red alga, Pyropia yezoensis.

A 2,158 bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of BPOs suggested that P. yezoensis and cyanobacteria were grouped in the same clade and separated from brown algae. Genomic Southern blot analysis suggested that PyBPO1 existed as a single copy per haploid genome. RT-PCR revealed that PyBPO1 was actively expressed in filamentous sporophytes but repressed in leafy gametophytes under normal growth conditions. High expression levels of PyBPO1 in sporophytes were observed when sporophytes were grown under gametophyte conditions, suggesting that preferential expression of PyBPO1 occurs during the sporophyte phase. BPO activity of cell-free extracts from sporophytes and gametophytes was examined by activity staining on native PAGE gel using o-dianisidine. One activity band was detected in sporophyte sample, but not in gametophyte sample. In addition, we found that bromide and iodide were effective substrate, but chloride was not. BPO activity was observed-likely in chloroplasts-when sporophyte cells were incubated with o-dianisidine and hydrogen peroxide. Cellular BPO staining showed the same halogen preference identified by in-gel BPO staining. Based on GS-MS analysis, bromoform was detected in medium containing sporophytes. Bromoform was not detected under dark culture conditions but was detected in the culture exposed to low light intensity (5 µmol m(-2) s(-1)) and increased under a moderate light intensity (30 µmol m(-2) s(-1)).

Involvement of microRNA in copper deficiency-induced repression of chloroplastic CuZn-superoxide dismutase genes in the moss Physcomitrella patens.

Superoxide dismutases (SODs) are metallo-enzymes that catalyze the dismutation of superoxide radicals. In Arabidopsis thaliana, the expression of CuZn-SOD in both the chloroplast and cytosol was reported to be down-regulated by microRNA398 (miR398) during growth on low copper. The moss Physcomitrella patens contains chloroplastic and cytosolic CuZn-SOD genes, but lacks miR398. From analysis of P. patens microRNA, miR1073 was predicted to target CuZn-SOD mRNAs. We noticed that two chloroplastic CuZn-SOD genes contain the miR1073 target sequence in the 3' untranslated region; however, the cytosolic isozyme genes lack this sequence. In this study, we investigated the involvement of miR1073 in the expression of CuZn-SOD genes in P. patens. When protonemata of P. patens were cultured on a copper-depleted medium, SOD activity and mRNA levels of chloroplastic CuZn-SODs were decreased markedly. In contrast, cytosolic CuZn-SODs showed little or no change in mRNA levels or SOD activity. The precursor transcript and the mature form of miR1073 were induced by copper deficiency. The chloroplastic CuZn-SOD (PpCSD1) mRNA was cleaved at the miR1073 target site under copper deficiency. These results suggest that miR1073 is involved in the down-regulation of PpCSD1 expression. In addition to PpCSD1 mRNA, antisense RNAs of PpCSD1 were also detected under normal conditions; however, under copper deficiency, they were cleaved within the open reading frame (ORF) region. The cleavage of sense PpCSD1 mRNA was also detected within the ORF region. Although only miR1073 exists in the database, it is presumed that RNA cleavage, other than that mediated by miR1073, is involved in the regulation of PpCSD1 expression.