An In Vivo Dual-Observation Method to Monitor Tumor Mass and Tumor-Surface Blood Vessels for Developing Anti-Angiogenesis Agents against Submillimeter Tumors.

Managing metastasis at the early stage and detecting and treating submillimeter tumors at early metastasis are crucial for improving cancer prognosis. Angiogenesis is a critical target for developing drugs to detect and inhibit submillimeter tumor growth; however, drug development remains challenging because there are no suitable models for observing the submillimeter tumor mass and the surrounding blood vessels in vivo. We have established a xenograft subcutaneous submillimeter tumor mouse model with HT-29-RFP by transplanting a single spheroid grown on radiation-crosslinked gelatin hydrogel microwells. Here, we developed an in vivo dual-observation method to observe the submillimeter tumor mass and tumor-surface blood vessels using this model. RFP was detected to observe the tumor mass, and a fluorescent angiography agent FITC-dextran was administered to observe blood vessels via stereoscopic fluorescence microscopy. The anti-angiogenesis agent regorafenib was used to confirm the usefulness of this method. This method effectively detected the submillimeter tumor mass and tumor-surface blood vessels in vivo. Regorafenib treatment revealed tumor growth inhibition and angiogenesis downregulation with reduced vascular extremities, segments, and meshes. Further, we confirmed that tumor-surface blood vessel areas monitored using in vivo dual-observation correlated with intratumoral blood vessel areas observed via fluorescence microscopy with frozen sections. In conclusion, this method would be useful in developing anti-angiogenesis agents against submillimeter tumors.

Preparation and Evaluation of Hydrogel Film Containing Tramadol for Reduction of Peripheral Neuropathic Pain.

To develop and assess new dosage forms for the alternative to existing oral medication for peripheral neuropathy, a hydrogel film in the skin patch formation containing tramadol hydrochloride (TRA), a water-soluble drug used as an analgesic, was prepared and evaluated. A hydrogel film composed of 20%(w/w) hydroxypropyl methylcellulose (HPMC) irradiated with electron beams had high transparency and elasticity similar to commercially available wound dressings and soft tissues, suggesting that it is a suitable substrate for TRA. The inclusion of TRA was enabled by immersing the HPMC hydrogel film in TRA aqueous solution. The release and skin permeation of TRA from TRA-containing hydrogel films differed depending on the electron beam dose. Moreover, the analgesic effects in mice were confirmed in a dose-dependent manner. This study demonstrated the usefulness of a hydrogel film containing TRA as a new dosage form alternative to the existing oral medication for peripheral neuropathy.

Synthesis of Small Peptide Nanogels Using Radiation Crosslinking as a Platform for Nano-Imaging Agents for Pancreatic Cancer Diagnosis.

Nanoparticle-based drug delivery systems (DDS) have been developed as effective diagnostic and low-dose imaging agents. Nano-imaging agents with particles greater than 100 nm are difficult to accumulate in pancreatic cancer cells, making high-intensity imaging of pancreatic cancer challenging. Peptides composed of histidine and glycine were designed and synthesized. Additionally, aqueous peptide solutions were irradiated with γ-rays to produce peptide nanogels with an average size of 25-53 nm. The mechanisms underlying radiation-mediated peptide crosslinking were investigated by simulating peptide particle formation based on rate constants. The rate constants for reactions between peptides and reactive species produced by water radiolysis were measured using pulse radiolysis. HGGGHGGGH (H9, H-histidine; G-glycine) particles exhibited a smaller size, as well as high formation yield, stability, and biodegradability. These particles were labeled with fluorescent dye to change their negative surface potential and enhance their accumulation in pancreatic cancer cells. Fluorescent-labeled H9 particles accumulated in PANC1 human pancreatic cancer cells, demonstrating that these particles are effective nano-imaging agents for intractable cancers.

A Radiation-Crosslinked Gelatin Hydrogel That Promotes Tissue Incorporation of an Expanded Polytetrafluoroethylene Vascular Graft in Rats.

A prosthetic vascular graft that induces perigraft tissue incorporation may effectively prevent serious sequelae such as seroma formation and infection. Radiation-crosslinked gelatin hydrogel (RXgel) mimics the chemical and physical properties of the in vivo extracellular matrix and may facilitate wound healing by promoting tissue organization. Fibroblasts cultured on RXgel actively migrated into the gel for up to 7 days. RXgels of three different degrees of hardness (Rx[10], soft; Rx[15], middle; Rx[20], hard) were prepared, and small disc-like samples of RXgels were implanted into rats. In vitro and in vivo results indicated that Rx[10] was too soft to coat vascular grafts. Thus, expanded polytetrafluoroethylene (ePTFE) vascular grafts coated with RXgel were developed using Rx[15] and Rx[20] gels, and ring-shaped slices of the graft were implanted into rats. Alpha-smooth muscle actin (αSMA) and type III collagen (Col-III) levels were detected by immunohistochemistry. Immunohistochemical staining for αSMA and Col-III demonstrated that RXgel-coated vascular grafts induced more granulation tissue than non-coated grafts on days 14 and 28 after implantation. RXgel-coated ePTFE vascular grafts may provide a solution for patients by reducing poor perigraft tissue incorporation.

Collagen hydrogels with controllable combined cues of elasticity and topography to regulate cellular processes.

The elasticity, topography, and chemical composition of cell culture substrates influence cell behavior. However, the cellular responses toin vivoextracellular matrix (ECM), a hydrogel of proteins (mainly collagen) and polysaccharides, remain unknown as there is no substrate that preserves the key features of native ECM. This study introduces novel collagen hydrogels that can combine elasticity, topography, and composition and reproduce the correlation between collagen concentration (C) and elastic modulus (E) in native ECM. A simple reagent-free method based on radiation-cross-linking altered ECM-derived collagen I and hydrolyzed collagen (gelatin or collagen peptide) solutions into hydrogels with tunable elastic moduli covering a broad range of soft tissues (E= 1-236 kPa) originating from the final collagen density in the hydrogels (C= 0.3%-14%) and precise microtopographies (⩾1 µm). The amino acid composition ratio was almost unchanged by this method, and the obtained collagen hydrogels maintained enzyme-mediated degradability. These collagen hydrogels enabled investigation of the responses of cell lines (fibroblasts, epithelial cells, and myoblasts) and primary cells (rat cardiomyocytes) to soft topographic cues such as thosein vivounder the positive correlation betweenCandE. These cells adhered directly to the collagen hydrogels and chose to stay atop or spontaneously migrate into them depending onE, that is, the density of the collagen network,C. We revealed that the cell morphology and actin cytoskeleton organization conformed to the topographic cues, even when they are as soft asin vivoECM. The stiffer microgrooves on collagen hydrogels aligned cells more effectively, except HeLa cells that underwent drastic changes in cell morphology. These collagen hydrogels may not only reducein vivoandin vitrocell behavioral disparity but also facilitate artificial ECM design to control cell function and fate for applications in tissue engineering and regenerative medicine.

Radiation Crosslinked Smart Peptide Nanoparticles: A New Platform for Tumor Imaging.

Nanoparticles have been employed to develop nanosensors and drug carriers that accumulate in tumors. Thus, it is necessary to control the particle size, surface potential, and biodegradability of these nanoparticles for effective tumor accumulation and safe medical application. In this study, to form a nanoparticle platform suitable for diagnostic and drug delivery system (DDS) applications, peptides composed of aromatic amino acid residues were designed and synthesized based on the radiation crosslinking mechanism of proteins. The peptide nanoparticles, which were produced by γ-ray irradiation, displayed a positive surface potential, maintained biodegradability, and were stable in water and phosphoric buffer solution during actual diagnosis. The surface potential of the peptide nanoparticles could be changed to negative by using a fluorescent labeling reagent, so that the fluorescent-labeled peptide nanoparticles were uptaken by HeLa cells. The radiation-crosslinked nanoparticles can be applied as a platform for tumor-targeting diagnostics and DDS therapy.

Ultra-small size gelatin nanogel as a blood brain barrier impermeable contrast agent for magnetic resonance imaging.

Magnetic Resonance Imaging (MRI) contrast agents with rapid renal excretion that do not penetrate the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCFB) are preferred for safer and low-risk diagnosis. Gadolinium (Gd)-conjugated nanoparticles have been proposed for use as contrast agents; however, the particle size must range between 1 to 7 nm to ensure rapid renal excretion. In this study, three types of gelatin, dissolved in water at varying concentrations of 0.1-2 wt.%, were irradiated with 5 kGy γ-rays at 25°C under aerated conditions to produce ultra-small gelatin nanogels having an average particle size ranging between 6 ± 2 to 21 ± 4 nm. Ultra-small Gd-coordinated gelatin nanogels (GdGN) suitable for use as MRI contrast agents were produced using 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS) and DOTA-butylamine as Gd ligand derivatives. Non-cytotoxicity and effective relaxivity of GdGN as a positive MRI contrast agent were verified using in vivo experiments. Rapid renal excretion of GdGN was observed in mice within 1 h with no accumulation in the liver. GdGN did not migrate across the BCFB in normal mice, thus emphasizing its safety as an MRI contrast agent. STATEMENT OF SIGNIFICANCE: The authors developed ultra-small sized gelatin nanogels as blood-brain-barrier impermeable contrast agents for magnetic resonance imaging (MRI). The authors used radiation crosslinking technique to ensure better integrity of the amino acids present in the gelatin nanogels while conjugating with gadolinium (Gd) to form gadolinium-coordinated gelatin nanogels (GdGN). The safety and efficacy of GdGN, as MRI contrast agents, were verified by in vivo studies. GdGN exhibited rapid renal excretion within 90 minutes and no passage across the barriers in the brain.

A simple method for production of hydrophilic, rigid, and sterilized multi-layer 3D integrated polydimethylsiloxane microfluidic chips.

Polydimethylsiloxane (PDMS) has many desirable features for microfluidics applications, particularly in diagnostics and pharmaceuticals, but its hydrophobicity and the lack of a practical method for bonding PDMS layers limit its use. Moreover, the flexibility of PDMS causes unwanted deformation during use in some applications. Here, we report a simple method for solving these problems simultaneously using an electron beam (EB) or γ-rays, which are commonly used for sterilizing medical products. Simply by applying EB or γ-ray irradiation to stacked PDMS layers, we can not only bond the interfaces between the layers by forming Si-O-Si covalent bonds but also achieve long-lasting hydrophilization and sterilization of the internal microchannels and chambers, prevent nonspecific adsorption and absorption of hydrophobic small molecules, and enhance the mechanical strength of the material by converting bulk PDMS into a Si-Ox-rich (where x is 3 or 4) structure though crosslinking. Unlike the one-at-a-time plasma process, EBs and γ-rays can penetrate through many stacked layers of PDMS sealed in their final package, enabling batch modification and bonding. The method requires no chemical crosslinkers, adhesive agents, or fillers; hence, it does not undermine the advantages of PDMS such as ease of molding in soft lithography, biocompatibility, and optical transparency. Furthermore, bonding is achieved with high-throughput yield because it occurs after re-adjustable alignment. We demonstrate that this method is applicable in the mass production of 3D integrated PDMS microfluidic chips with some glass-like properties as well as for 3D structures with complex shapes that are difficult to fabricate with plastic or glass.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

On-line rapid purification of [13N]N2 gas for visualization of nitrogen fixation and translocation in nodulated soybean.

Accurate analysis of N fixation in leguminous crops requires determination of N utilization within an intact plant; however, most approaches require tissue disassembly. We developed a simple and rapid technique to generate high-purity and high-yield [13N]N2 gas and obtained real-time images of N fixation in an intact soybean plant. The purification efficiency was ∼81.6% after decay correction. Our method provides accurate signals of N fixation and allows free changes to the tracer gas composition to suit different experimental designs.

Effect of Gamma Ray Irradiation on Friction Property of Poly(vinyl alcohol) Cast-Drying on Freeze-Thawed Hybrid Gel.

Poly(vinyl alcohol) (PVA) is a biocompatible polymer with low toxicity. It is possible to prepare physically cross-linked PVA gels having hydrogen bonds without using a cross-linking agent. The newly reported physically cross-linked PVA cast-drying (CD) on freeze-thawed (FT) hybrid gel has an excellent friction property, which is expected to be applied as a candidate material for artificial cartilage. Gamma ray sterilization for clinical applications usually causes additional chemical cross-linking and changes physical properties of gels. In this study, CD on FT hybrid gels were irradiated using gamma rays at a different dose rate and irradiance. The results showed the optimized irradiation conditions for gamma irradiated gels to retain excellent friction characteristics.

[Evaluation of Three Dimensional Dose Distributions by Using Polymer Gel Dosimeters Based on HPC Gel Sheets].

We have proposed a novel polymer gel dosimeter containing of 2-hydroxyethyl methacrylate (HEMA), nonaethylene glycol dimethacrylate (9G), and tetrakis (hydroxymethyl) phosphonium chloride (THPC) with radiation-crosslinked hydroxypropyl cellulose (HPC) gel sheet. The transparent sheet-type dosimeters became white and cloudy by irradiation with gamma-rays and heavy ions such as He ions (150 MeV/u), C ions (290 MeV/u), Fe ions (500 MeV/u). The cloudiness increased with increasing dose. The cloudiness distribution with the sheet-type dosimeter was obtained by using a flatbed scanner to evaluate the dose distribution. Recently, we prepared a three-dimensional dosimeter by putting the gel sheets on top of another in the glass vessel. Three-dimensional dose distribution of the dosimeter irradiated with C ions was evaluated by the reconstruction of the data of each layer.

Radiation-Induced Chemical Reactions in Hydrogel of Hydroxypropyl Cellulose (HPC): A Pulse Radiolysis Study.

We performed studies on pulse radiolysis of highly transparent and shape-stable hydrogels of hydroxypropyl cellulose (HPC) that were prepared using a radiation-crosslinking technique. Several fundamental aspects of radiation-induced chemical reactions in the hydrogels were investigated. With radiation doses less than 1 kGy, degradation of the HPC matrix was not observed. The rate constants of the HPC composing the matrix, with two water decomposition radicals [hydroxyl radical (•OH) and hydrated electron ([Formula: see text])] in the gels, were determined to be 4.5 × 109 and 1.8 × 107 M-1 s-1, respectively. Direct ionization of HPC in the matrix slightly increased the initial yield of [Formula: see text], but the additionally produced amount of [Formula: see text] disappeared immediately within 200 ps, indicating fast recombination of [Formula: see text] with hole radicals on HPC or on surrounding hydration water molecules. Reactions of [Formula: see text] with nitrous oxide (N2O) and nitromethane (CH3NO2) were also examined. Decay of [Formula: see text] due to scavenging by N2O and CH3NO2 were both slower in hydrogels than in aqueous solutions, showing slower diffusions of the reactants in the gel matrix. The degree of decrease in the decay rate was more effective for N2O than for CH3NO2, revealing lower solubility of N2O in gel than in water. It is known that in viscous solvents, such as ethylene glycol, CH3NO2 exhibits a transient effect, which is a fast reaction over the contact distance of reactants and occurs without diffusions of reactants. However, such an effect was not observed in the hydrogel used in the current study. In addition, the initial yield of [Formula: see text], which is affected by the amount of the scavenged precursor of [Formula: see text], in hydrogel containing N2O was slightly higher than that in water containing N2O, and the same tendency was found for CH3NO2.

Enhancement of beam pulse controllability for a single-pulse formation system of a cyclotron.

The single-pulse formation technique using a beam chopping system consisting of two types of high-voltage beam kickers was improved to enhance the quality and intensity of the single-pulse beam with a pulse interval over 1 µs at the Japan Atomic Energy Agency cyclotron facility. A contamination rate of neighboring beam bunches in the single-pulse beam was reduced to less than 0.1%. Long-term purification of the single pulse beam was guaranteed by the well-controlled magnetic field stabilization system for the cyclotron magnet. Reduction of the multi-turn extraction number for suppressing the neighboring beam bunch contamination was achieved by restriction of a beam phase width and precise optimization of a particle acceleration phase. In addition, the single-pulse beam intensity was increased by a factor of two or more by a combination of two types of beam bunchers using sinusoidal and saw-tooth voltage waveforms. Provision of the high quality intense single-pulse beam contributed to improve the accuracy of experiments for investigation of scintillation light time-profile and for neutron energy measurement by a time-of-flight method.

System for measuring temporal profiles of scintillation at high and different linear energy transfers by using pulsed ion beams.

We have developed a system for measuring the temporal profiles of scintillation at high linear energy transfer (LET) by using pulsed ion beams from a cyclotron. The half width at half maximum time resolution was estimated to be 1.5-2.2 ns, which we attributed mainly to the duration of the pulsed ion beam and timing jitter between the trigger signal and the arrival of the ion pulse. The temporal profiles of scintillation of BaF2 at different LETs were successfully observed. These results indicate that the proposed system is a powerful tool for analyzing the LET effects in temporal profiles of scintillation.

Mechanism of radiation-induced reactions in aqueous solution of coumarin-3-carboxylic acid: effects of concentration, gas and additive on fluorescent product yield.

The radiation-induced reactions of a water-soluble coumarin derivative, coumarin-3-carboxyl acid (C3CA), have been investigated in aqueous solutions by pulse radiolysis with a 35 MeV electron beam, final product analysis following (60)Co γ-irradiations and deterministic model simulations. Pulse radiolysis revealed that C3CA reacted with both hydroxyl radicals ((•)OH) and hydrated electrons (e(-) (aq)) with near diffusion-controlled rate constants of 6.8 × 10(9) and 2.1 × 10(10) M(-1) s(-1), respectively. The reactivity of C3CA towards O(2)(• -) was not confirmed by pulse radiolysis. Production of the fluorescent molecule, 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), was confirmed by final product analysis with a fluorescence spectrometer coupled to a high performance liquid chromatography (HPLC) system. Production yields of 7OH-C3CA following (60)Co γ-irradiations depended on the irradiation conditions and ranged from 0.025 to 0.18 (100 eV) (-1). Yield varied with saturating gas, additive and C3CA concentration, implying the presence of at least two pathways capable of providing 7OH-C3CA as a stable product following the scavenging reaction of C3CA with (•)OH, including a peroxidation/elimination sequence and a disproportionation pathway. A reaction mechanism for the two pathways was proposed and incorporated into a deterministic simulation, showing that the mechanism can explain experimentally measured 7OH-C3CA yields with a constant conversion factor of 4.7% from (•)OH scavenging to 7OH-C3CA production, unless t-BuOH was added.

Estimation of yields of hydroxyl radicals in water under various energy heavy ions.

This article reports the determination of yields of OH (hydroxyl) radicals in water irradiated with helium, carbon, neon and argon ions ranging from 2 to 18 MeV/nucleon. The yields of the OH radicals depend on the atomic number and energy of the incident ion and the reaction time just after the irradiation based on the track structure theory. The yields of the OH radicals estimated by analyzing the yields of the irradiation products from phenol were at almost 0 to 3.1 per 100 eV absorbed energy on a time scale from 0.75 to 300 ns and were lower than the corresponding ones after exposure to low-LET radiation. The yields of OH radicals decreased with decreasing specific energy for each ion, with increasing atomic number of each ion at a similar specific energy, and with the average reaction time after irradiation. In addition, Monte Carlo simulations were conducted and compared with the OH radical yields obtained experimentally.

Heavy-ion microbeam system at JAEA-Takasaki for microbeam biology.

Research concerning cellular responses to low dose irradiation, radiation-induced bystander effects, and the biological track structure of charged particles has recently received particular attention in the field of radiation biology. Target irradiation employing a microbeam represents a useful means of advancing this research by obviating some of the disadvantages associated with the conventional irradiation strategies. The heavy-ion microbeam system at JAEA-Takasaki, which was planned in 1987 and started in the early 1990's, can provide target irradiation of heavy charged particles to biological material at atmospheric pressure using a minimum beam size 5 mum in diameter. A variety of biological material has been irradiated using this microbeam system including cultured mammalian and higher plant cells, isolated fibers of mouse skeletal muscle, silkworm (Bombyx mori) embryos and larvae, Arabidopsis thaliana roots, and the nematode Caenorhabditis elegans. The system can be applied to the investigation of mechanisms within biological organisms not only in the context of radiation biology, but also in the fields of general biology such as physiology, developmental biology and neurobiology, and should help to establish and contribute to the field of "microbeam biology".

Yield of OH radicals in water under high-density energy deposition by heavy-ion irradiation.

The purpose of the present study was to evaluate the dependence of the OH radical yield on the atomic number and the energy of the heavy ions to understand chemical reactions of aqueous solutions. The total yields of oxidized products from phenol in water increased superlinearly as the incident energy increased from 5 MeV/nucleon to 18 MeV/nucleon for carbon and neon ions. The radiolytic yields of OH radicals produced by the ions were determined by analyzing the relationships of the oxidation yields of phenol to the incident energies up to 18 MeV/nucleon for ions in the range of LET from 110 eV/nm to 550 eV/nm and from 320 eV/nm to 1100 eV/nm for carbon and neon ions, respectively. The yields of the OH radicals increased with the specific energy for the same kind of ion and decreased with the atomic number for different ions used at the same specific energy.

Application of electron beam for the reduction of PCDD/F emission from municipal solid waste incinerators.

The electron-beam technology was applied to reduce the emission of polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) in a flue gas of 1000 m(3)N/h from the municipal solid waste incinerator (MSWI) at a temperature of 200 degrees C. More than 90% decomposition of PCDD/Fs was obtained using an electron accelerator at a dose of 14 kGy. The decomposition was initiated through reactions with OH radicals produced by the irradiation of flue gases, followed by oxidation such as the ring cleavage of the aromatic ring, the dissociation of ether bond, and dechlorination. The cost analysis estimated that the electron-beam system can cut the annualized cost by approximately 50% for the treatment of PCDD/Fs in a pre-dusted MSWI flue gas as compared with a bag-filter system when operating on electricity generated from an incineration. Electron-beam technology is an economically and technologically useful method for reducing PCDD/Fs in an incineration flue gas.