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.

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.

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.

[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.

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.

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.

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.

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.

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.

Preparation of stable insulin-loaded nanospheres of poly(ethylene glycol) macromers and N-isopropyl acrylamide.

A series of nanospheres composed of temperature-sensitive poly(N-isopropylacrylamide), poly(ethylene glycol) 400 dimethacrylate, and poly(ethylene glycol) 1000 methacrylate was prepared by a thermally-initiated free radical dispersion polymerization method. Insulin was loaded into the nanoparticles by equilibrium partitioning. The loading capacity of insulin into the nanoparticles was 2.1% (2.1 mg insulin/100 mg nanoparticles). The stability of the loaded insulin at elevated temperatures was investigated by reverse phase high pressure liquid chromatography. The nanoparticles were able to protect the loaded insulin, as more than 80% of the loaded insulin could still be detected compared to 0% for the control (0.1% insulin solution in PBS) when heated to 80 degrees C for 5 h. The stability of the loaded insulin at high shear stress (289 1/s) was also investigated. No significant loss of insulin was detected both from nanoparticles loaded with insulin sample and the control (0.1% insulin solution in PBS). The results showed that shear stress alone did not have a major effect on insulin denaturation. The ability of the nanoparticles to protect the insulin from high temperature and high shear stress made the system a good candidate as a carrier for insulin for fluidized bed coating technology.

Tumor accumulation of gadolinium in lipid-nanoparticles intravenously injected for neutron-capture therapy of cancer.

Gadolinium-incorporating lipid-nanoemulsions (Gd-nanoLE) for neutron-capture therapy were prepared. As a more convenient administration route than the intraperitoneal (i.p.) injection previously reported, the intravenous (i.v.) injections in tumor-bearing hamsters were carried out at an administration volume of 1 ml, which was the maximum tolerable injection volume of an i.v. injection and half that of an i.p. injection. When the standard-Gd-nanoLE of 1.5 mgGd/ml was administered, the absolute bioavailability in the i.p. injection was 57%, probably resulting from incomplete absorption from the peritoneal cavity into the blood stream. The biodistribution data revealed that the i.v. injection had three advantages over the i.p. injection, namely, a faster and higher accumulation of Gd-nano LE, and a more extended retention time in the tumor. Two i.v. injections of the standard-Gd-nanoLE with a 24h interval doubled the tumor accumulation of Gd, resulting in 49.7 microg Gd/g wet tumor 12h after administration. By using a twofold Gd-enriched formulation (High-Gd-nanoLE) of 3.0 mgGd/ml in the repeated administration schedule, the accumulation was doubled again, reaching 101 microg Gd/g wet tumor. This level was comparable to the maximum level in the single i.p. injection previously reported. These results demonstrated that i.v. injection could be an alternative to i.p. injection as an administration route.

In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer.

The accumulation of gadolinium loaded as gadopentetic acid (Gd-DTPA) in chitosan nanoparticles (Gd-nanoCPs), which were designed for gadolinium neutron-capture therapy (Gd-NCT) for cancer, was evaluated in vitro in cultured cells. Using L929 fibroblast cells, the Gd accumulation for 12 h at 37 degrees C was investigated at Gd concentrations lower than 40 ppm. The accumulation leveled above 20 ppm and reached 18.0+/-2.7 (mean+/-S.D.) microg Gd/10(6) cells at 40 ppm. Furthermore, the corresponding accumulations in B16F10 melanoma cells and SCC-VII squamous cell carcinoma, which were used in the previous Gd-NCT trials in vivo, were 27.1+/-2.9 and 59.8+/-9.8 microg Gd/10(6) cells, respectively, hence explaining the superior growth-suppression in the in vivo trials using SCC-VII cells. The accumulation of Gd-nanoCPs in these cells was 100-200 times higher in comparison to dimeglumine gadopentetate aqueous solution (Magnevist), a magnetic resonance imaging contrast agent. The endocytic uptake of Gd-nanoCPs, strongly holding Gd-DTPA, was suggested from transmission electron microscopy and comparative studies at 4 degrees C and with the solution system. These findings indicated that Gd-nanoCPs had a high affinity to the cells, probably contributing to the long retention of Gd in tumor tissue and leading to the significant suppression of tumor growth in the in vivo studies that were previously reported.

Inhibitory action of emulsified sulfoquinovosyl acylglycerol on mammalian DNA polymerases.

We reported previously that sulfoquinovosyl diacylglycerol and sulfoquinovosyl monoacylglycerol (SQDG/SQMG) are potent inhibitors of mammalian DNA polymerases and DNA topoisomerase II, and can be potent immunosuppressive agents and anticancer chemotherapy agents [Matsumoto, Y., Sahara, H., Fujita T., Shimozawa, K., Takenouchi, M., Torigoe, T., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., et al., An Immunosuppressive Effect by Synthetic Sulfonolipids Deduced from Sulfonoquinovosyl Diacylglycerols of Sea Urchin, Transplantation 74, 261-267 (2002); Sahara, H., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., Ohtani, S., Ishikawa, M., Mizushina, Y., Ohta, K., Shimozawa, K., et al., Anti-tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols, Jpn. J. Cancer Res. 93, 85-92 (2002)]. In those experiments, the in vivo effectiveness greatly depended on the degree of water solubility of SQDG/SQMG. In the present work, we studied the emulsification of SQDG/SQMG in terms of their use in in vivo experiments. Lipid emulsions containing SQDG/SQMG (oil-in-water emulsions) in which the particle size was smaller than 100 nm were designed and synthesized, and then the biochemical modes of emulsified SQDG/SQMG were studied in comparison with those of SQDG/SQMG solubilized by DMSO. Emulsified SQDG/SQMG are also selective mammalian DNA polymerase inhibitors and potent antineoplastic agents but do not inhibit the DNA topoisomerase II activity. The growth inhibition effect of emulsified SQMG to NUGC-3 cancer cells was twofold stronger than DMSO-soluble SQMG (69 and 151 microM, respectively). From these results, the properties of lipid emulsions containing SQDG/SQMG and their possible use in in vivo experiments including clinical use are discussed.

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.

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.

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.