Boron neutron capture therapy (BNCT) as a new approach for clear cell sarcoma (CCS) treatment: Trial using a lung metastasis model of CCS.

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In the present study, we established a lung metastasis animal model of CCS and investigated the therapeutic effect of boron neutron capture therapy (BNCT) using p-borono-L-phenylalanine (L-BPA). Biodistribution data revealed tumor-selective accumulation of (10)B. Unlike conventional gamma-ray irradiation, BNCT significantly suppressed tumor growth without damaging normal tissues, suggesting that it may be a potential new therapeutic option to treat CCS lung metastases.

Potential of boron neutron capture therapy (BNCT) for malignant peripheral nerve sheath tumors (MPNST).

Malignant peripheral nerve sheath tumors (MPNST) are relatively rare neoplasms with poor prognosis. At present there is no effective treatment for MPNST other than surgical resection. Nonetheless, the anti-tumor effect of boron neutron capture therapy (BNCT) was recently demonstrated in two patients with MPNST. Subsequently, tumor-bearing nude mice subcutaneously transplanted with a human MPNST cell line were injected with p-borono-L-phenylalanine (L-BPA) and subjected to BNCT. Pathological studies then revealed that the MPNST cells were selectively destroyed by BNCT.

Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy. III: racemization studies and water-based synthesis of histidine-containing peptides.

In this study, we describe the first aqueous microwave-assisted synthesis of histidine-containing peptides in high purity and with low racemization. We have previously shown the effectiveness of our synthesis methodology for peptides including difficult sequences using water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles. It is an organic solvent-free, environmentally friendly method for chemical peptide synthesis. Here, we studied the racemization of histidine during an aqueous-based coupling reaction with microwave irradiation. Under our microwave-assisted protocol using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, the coupling reaction can be efficiently performed with low levels of racemization of histidine. Application of this water-based microwave-assisted protocol with water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles led to the successful synthesis of the histidine-containing hexapeptide neuropeptide W-30 (10-15), Tyr-His-Thr-Val-Gly-Arg-NH2, in high yield and with greatly reduced histidine racemization.

Boron neutron capture therapy as new treatment for clear cell sarcoma: trial on different animal model.

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In our previous study, the tumor disappeared under boron neutron capture therapy (BNCT) on subcutaneously-transplanted CCS-bearing animals. In the present study, the tumor disappeared under this therapy on model mice intramuscularly implanted with three different human CCS cells. BNCT led to the suppression of tumor-growth in each of the different model mice, suggesting its potentiality as an alternative to, or integrative option for, the treatment of CCS.

Gadolinium-loaded chitosan nanoparticles for neutron-capture therapy: Influence of micrometric properties of the nanoparticles on tumor-killing effect.

As a nanoparticulate device for controlled delivery of Gd in NCT, the authors have developed gadolinium-loaded chitosan nanoparticles (Gd-nanoCPs). In the present study, influence of micrometric properties such as particle size, particle-surface charge and Gd content of Gd-nanoCPs on tumor-killing effect by Gd-NCT was investigated with Gd-nanoCPs. Two types of Gd-nanoCPs with different mean particle size, zeta potential and Gd-content (Gd-nanoCP-400; 391nm, 28mV, 9wt% and Gd-nanoCP-200; 214nm, 19mV, 24wt%) could be prepared by using chitosans with different molecular weights. Gd-nanoCPs incorporating 1.2mg of natural Gd were injected intratumorally once or twice to mice subcutaneously-bearing B16F10 melanoma. Eight hours after the last administration, thermal neutron was irradiated to tumor region of the mice. Remarkable tumor-growth was observed in both hot and cold control groups. In contrast, Gd-NCT groups showed significant tumor-growth suppression effect, though their efficacy was found to depend on the micrometric properties of Gd-nanoCPs. In particular, the Gd-nanoCP-200 exhibited stronger tumor-killing effect than the Gd-nanoCP-400 at the same Gd dose and it was still similar to Gd-nanoCP-400 in tumor-growth suppressing effect even at the half of Gd dose of Gd-nanoCP-400. This significance in tumor-killing effect would be ascribed from a higher Gd retention in the tumor tissue and an improved distribution of Gd with intratumorally administered Gd-nanoCP-200. Indeed, the Gd concentration in tumor tissue at the time corresponding to the onset of thermal neutron irradiation was determined to be significantly higher in Gd-nanoCP-200, compared with Gd-nanoCP-400. These results demonstrated that appropriate modification of Gd-nanoCPs in micrometric properties would be an effective way to improve the retention of Gd in the tumor tissue after intratumoral injection, leading to the enhanced tumor-killing effect in Gd-NCT.

Aqueous microwave-assisted solid-phase peptide synthesis using fmoc strategy. II. Racemization studies and water based synthesis of cysteine-containing peptides.

We have developed a microwave (MW)-assisted peptide synthesis using Fmoc-amino acid nanoparticles in water previously. It is an organic solvent-free, environmentally friendly method for peptide synthesis. In this study, we have investigated the racemization of cysteine during an aqueous based coupling reaction with MW irradiation. Under our MW-assisted protocol using WSCI and DMTMM, the coupling reaction can be performed with low levels of racemization of cysteine. We also demonstrated the synthesis of the nonapeptide oxytocin analogue, Cys(Acm)-Tyr-Ile-Gln-Asn- Cys(Acm)-Pro-Leu-Gly-NH2 using our water based MW-assisted protocol with Fmoc-amino acid nanoparticles.

Boron neutron capture therapy (BNCT) selectively destroys human clear cell sarcoma in mouse model.

Clear cell sarcoma of tendons and aponeuroses (CCS) is a rare malignant tumor with no effective treatment. This study demonstrates the efficacy of BNCT with the use of human CCS-bearing nude mice. Groups A and C were administered saline, and groups B and D were injected with p-borono-L-phenylalanine-fructose complex. Groups C and D were then irradiated with thermal neutrons. The tumors in only group D disappeared, demonstrating that BNCT is a potentially new option for the treatment of human CCS.

Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy: in-water synthesis of "difficult sequences".

A microwave assisted peptide synthesis in water using nanosized Fmoc-amino acids was developed. 5, 7, and 10 mer peptides (Leu-enkephalinamide, dermorphinamide, and a typical difficult sequence, ACP (65-74) peptide) were successfully synthesized in water according to Fmoc chemistry using water-dispersible nanoparticles with microwave irradiation.

Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles.

Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids.

Peptide synthesis 'in water' by a solution-phase method using water-dispersible nanoparticle Boc-amino acid.

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in-water solution-phase peptide synthesis method using Boc-amino acids. It is based on a coupling reaction utilizing suspended water-dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity.

Direct measurement of interactions between stimulation-responsive drug delivery vehicles and artificial mucin layers by colloid probe atomic force microscopy.

A novel thermo- and pH-sensitive nanogel particle, which is a core-shell structured particle with a poly(N-isopropylacrylamide) (p(NIPAAm)) hydrogel core and a poly(ethylene glycol) monomethacrylate grafted poly(methacrylic acid) (p(MMA-g-EG)) shell, is of interest as a vehicle for the controlled release of peptide drugs. The interactions between such nanogel particles and artificial mucin layers during both approach and separation were successfully measured by using colloid probe atomic force microscopy (AFM) under various compression forces, scan velocities, and pH values. While the magnitudes of the compression forces and scan velocities did not affect the interactions during the approach process, the adhesive force during the separation process increased with these parameters. The pH values significantly influenced the interactions between the nanogel particles and a mucin layer. A large steric repulsive force and a long-range adhesive force were measured at neutral pH due to the swollen p(MMA-g-EG) shell. On the other hand, at low pH values, the steric repulsive force disappeared and a short-range adhesive force was detected, which resulted from the collapse of the shell layer. The nanogel particles possessed a pH response that was sufficient to protect the incorporated peptide drug under the harsh acidic conditions in the stomach and to effectively adhere to the mucin layer of the small intestine, where the pH is neutral. The relationships among the nanogel particle-mucin layer interactions, pH conditions, scan velocities, and compression forces were systemically investigated and discussed.

Chemical groups that adhere to the surfaces of living malignant cells.

PURPOSE: We determined the adhesion of particles with phenyl, carboxylic acid (COOH), amine, dialkyl phosphonate, ester, and hydroxyl groups to malignant and nonmalignant cells, in order to better design drug delivery systems (DDS) for malignant cells. METHODS: Living mouse melanoma skin (B16F10) and noncancerous mouse fibroblast (L929) cells, and an Atomic Force Microscope were used to determine the adhesion strengths. RESULTS: The measurement of the particles against B16F10 cells showed that COOH had the highest average maximum adhesion force () and a large standard deviation (std), and phenyl had the lowest and a lower std. The high and std suggested that COOH was binding the strongest to malignant cells, and to groups overexpressed on malignant cells. In the case of L929 cells, of phenyl and COOH were higher and lower, respectively, than those of the B16F10 cells. Additionally, Phenyl and COOH gave a lower std than that for the B16F10 cells. These results suggest that the lower binding of COOH to the nonmalignant cells was due to the lower number of groups that were overexpressed in the malignant cells. CONCLUSIONS: Our results suggest that COOH is the best group for malignant cell targeting DDS systems.

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.

[Design of nanohydrogel-incorporated microcapsules for appropriate controlled-release of peptide drugs].

Biologically active peptides for therapeutic use have relatively short half-lives in general, requiring appropriate controlled-release systems for better therapy. Controlled release of peptides is, however, not as easy as that of conventional drugs because their large molecular size is much more dramatic in hindering the diffusion and release from polymeric devices. From this perspective, we have been developing two types of microcapsular devices containing new acrylate-based nanogels with a specific solute-permeability for delayed- or thermosensitive-release of peptide drugs. The microcapsule preparation was accomplished by an air suspension coating process. A nanogel-particle of acrylic terpolymer, ethyl acrylate-methyl methacrylate-2-hydroxyethyl methacrylate, was newly synthesized by emulsion polymerization to construct delayed-release microcapsules. By spray-coating the insulin-loaded lactose particles with the acrylic terpolymers, microcapsules showing a pH-independent delayed-release profile can be obtained. Oral administration of the microcapsules with the lag time of 6 hours to beagle dogs resulted in significantly reduced blood glucose concentration, leading to colon-specific insulin delivery with pharmacological availability of 5%. Meanwhile, poly(N-isopropylcarylamide) (p(NIPAAm)) nanogel-particles with a reversible temperature-dependent swelling property were prepared by dispersion polymerization to fabricate microcapsular membranes with thermosensitively changeable permeability. The microcapsules constructed by coating of drug-loaded CaCO(3) particles with a blend mixture of the p(NIPAAm) nanogels and ethylcellulose pseudo-latex exhibited an 'on-off' positively thermosensitive drug-release; the release rate was remarkably enhanced at higher temperatures possibly due to the formation of voids through the shrinkage of p(NIPAAm) nanogels in the membrane. A possible application of this type of microcapsules can be found in externally temperature-activated pulsatile peptide delivery.

Formulation considerations of gadolinium lipid nanoemulsion for intravenous delivery to tumors in neutron-capture therapy.

The effects of the formulation and particle composition of gadolinium (Gd)-containing lipid nanoemulsion (Gd-nanoLE) on the biodistribution of Gd after its intravenous (IV) injection in D(1)-179 melanoma-bearing hamsters were evaluated for its application in cancer neutron-capture therapy. Gd-nanoLEs whose particles had an oily core (soybean oil, ethyl oleate, lipiodol, or triolein) and a surface layer of hydrogenated phosphatidylcholine, gadolinium-diethyl-enetriaminepentaacetic acid-distearylamide, and a cosurfactant (Myrj 53, Brij 700, or HCO-60) were prepared by a thin-layer hydration-sonication method. Biodistribution data revealed that Brij 700 and HCO-60 prolonged the retention of Gd in the blood and enhanced its accumulation in tumors. Among the core components employed, soybean oil yielded the highest Gd concentration in the blood and tumor and the lowest in the liver and spleen. Gd-nanoLEs with a Gd content of 1.5-4.5 mg/ml could be formulated by using HCO-60 and soybean oil at a constant oil-to-water ratio, and by enriching Gd in the surface layer with the particle size maintained below 100 nm. When each Gd-nanoLE was IV injected once or twice at a 24-h interval, the Gd concentration in the tumor correlated well with the total dose of Gd, and it reached a maximum of 189 microg/g wet tumor. This maximum Gd level was greater than the limit required for significantly suppressing tumor growth in neutron-capture therapy.

Effect of the cell type and cell density on the binding of living cells to a silica particle: an atomic force microscope study.

We used the atomic force microscope to study how the cell type and the density of cells adsorbed at a substrate can affect the adhesion between a living cell and a model drug delivery system (DDS) carrier nano-particle. We used three different anchorage-dependent cells, i.e., a living mouse fibroblast cell (L929), a living human colon cancer cell (Caco2), and a living mouse malignant melanoma cell (B16F10). For the DDS model nano-particle, we used a silica colloid. In order to correlate the adhesion force with the cell types, the growth curve of the cells were determined with a haemocytometer. The shapes of the cells at the different stages were monitored by light microscopy, and the morphology of their surfaces obtained by tapping mode atomic force microscopy. Force measurements showed that the Caco2 cell bound little to a silica particle, regardless of the cell density. The L929 cell bound well to a silica particle for low and high cell densities. The B16F10 cell bound little to a silica particle for low cell densities, but bound well for high cell densities. AFM images showed that the L929 cell did not contain folds. The B16F10 cells, however, displayed folds in the cell surface for low cell densities, but no folds in the cell for high cell densities. As literature also reported that the Caco2 cell contains folds, these results suggested that cells with folds showed less adhesion to a silica particle than cells without folds. The presence of folds in the cell presumably decreased the number of sites on the cell that could hydrogen bond or undergo van der Waals binding with the silanol groups of the silica particle.

Gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres for gadolinium neutron-capture therapy.

In order to provide a suitable device that would contain water-soluble drugs, highly water-soluble gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres (CMS-Gd-DTPA) were prepared by the emulsion method using glutaraldehyde as a cross-linker and Span 80 as a surfactant for gadolinium neutron-capture therapy of cancer. The gadolinium content and the mass median diameter of CMS-Gd-DTPA were estimated. The size and morphology of the CMS-Gd-DTPA were strongly influenced by the initial applied weight ratio of Gd-DTPA:chitosan. FTIR spectra showed that the electrostatic interaction between chitosan and Gd-DTPA accelerated the formation of gadolinium-enriched chitosan microspheres. Sufficient amounts of glutaraldehyde and Span 80 were necessary for producing discrete CMS-Gd-DTPA. The CMS-Gd-DTPA having a mass median diameter 11.7microm and 11.6% of gadolinium could be used in Gd-NCT following intratumoral injection.

Short-term delayed-release microcapsules spraycoated with acrylic terpolymers.

A series of poly(ethyl acrylate (EA)/methyl methacrylate (MMA)/2-hydroxyethyl methacrylate (HEMA)) lattices were synthesized to prepare short-term delayed-release microcapsules by employing the Wurster coating process. Latex with a HEMA molar fraction exceeding 60% could not be synthesized as an aqueous suspension due to latex particle precipitation. The effects of monomer composition on the particle size of latex and the water-uptake and glass transition temperature (T(g)) of cast films were investigated. Lattices whose T(g) ranged from 40 to 80 degrees C were used to prepare the microcapsules. Most of the lattices exhibited excellent process performance while coating particles that were smaller than 100 microm: the product yields were 85.1-90.6% and the mean particle sizes were 82-85 microm. However, since the lattices with high molar ratios of EA and HEMA were highly hydrophilic and strongly adhesive, the core particles in the coating were severely agglomerated. The microcapsules coated with lattices whose HEMA molar fractions were higher than 50% were unable to retard the release of carbazochrome sodium sulfonate, a water-soluble model drug, during the initial 0.5 min. Poly(EA/MMA/HEMA) with a molar ratio of 9:9:10 appeared to be suitable for the preparation of short-term delayed-release microcapsules by the Wurster coating process.

Mechanisms and kinetics of trisodium 2-hydroxy-1,1'-azonaphthalene-3,4',6-trisulfonate adsorption onto chitosan.

Chitosan, a naturally abundant biopolymer, has widely been studied for metal adsorption from various solutions, but the extension of chitosan as an adsorbent to remove organic substances from water and wastewater has seldom been explored. In this study, the adsorption of an azo dye, trisodium 2-hydroxy-1,1'-azonaphthalene-3,4',6-trisulfonate (1), from aqueous solution onto the various degrees of deacetylated chitosan has been investigated. Equilibrium studies have been carried out to determine the capacity of chitosan for dye. The experimental data were analyzed using two isotherm correlations, namely, Langmuir and Freundlich equations. The linear correlation coefficients were determined for each isotherm and the Langmuir provided the best fit. The experimental adsorption isotherms were perfectly reproduced in the simulated data obtained from numerical analysis on the basis of the Langmuir model and the isotherm constants. Adsorption of (1) onto the chitosan flakes was found to be strongly depending on degrees of deacetylation in chitosan and temperatures. Significant amounts of (1) were adsorbed by chitosan 8B (higher degree of deacetylated chitosan), but the adsorption capacity was reduced remarkably with increasing solution temperatures. Thermodynamic parameters such as change in free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) were also determined. In addition, kinetic study indicated that the adsorption process mechanisms were both transport- and attachment-limited.

Effects of DNA polymerase inhibitory and antitumor activities of lipase-hydrolyzed glycolipid fractions from spinach.

We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG), monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greene's melanoma, the fraction containing SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.