Evaluation of the Safety and Gastrointestinal Migration of Guanidinylated Chitosan after Oral Administration to Rats.

Arginine-rich membrane-permeable peptides (APPs) can be delivered to cells by forming complexes with various membrane-impermeable bioactive molecules such as proteins. We recently reported on the preparation of guanidinylated chitosan (GCS) that mimics arginine peptides, using chitosan, a naturally occurring cationic polysaccharide, and confirmed that it enhances protein permeability in an in vitro cell system. However, studies on the in vivo safety of GCS are not available. To address this, we evaluated the in vivo safety of GCS and its translocation into the gastrointestinal tract in rats after a single oral administration of an excessive dose (500 mg/kg) and observed changes in body weight, major organ weights, and organ tissue sections for periods of up to 2 weeks. The results indicated that GCS causes no deleterious effects. The results of an oral administration of rhodamine-labeled chitosan and an evaluation of its migration in the gastrointestinal tract suggested that the disappearance of rhodamine-labeled GCS from the body appeared to be slower than that of the non-dose group and pre-guanidinylated chitosan due to its mucoadhesive properties. In the future, we plan to investigate the use of GCS to improve absorption using Class III and IV drugs, which are poorly water-soluble as well as poorly membrane-permeable.

Production of chitin nanoparticles by bottom-up approach from alkaline chitin solution.

Most of the series of nanochitins have been produced by the break-down process. In this study, chitin nanoparticles were prepared by a bottom-up process. Chitin was treated with sodium hydroxide to obtain an alkaline chitin aqueous solution. The alkaline chitin was regenerated by neutralization and then vigorously stirred to obtain chitin nanoparticles. The average particle size of the chitin nanoparticles was 7 nm. The individual particles were stably dispersed in water. Chitin nanoparticles had lower crystallinity than the raw material chitin and the surface of the chitin nanoparticles regenerated in water were presumed to be hydrophilic. The low crystallinity and the high hydrophilicity of the surface contributed to the high dispersibility of the chitin nanoparticles in water. Chitin nanoparticles had higher heat resistance than the raw material chitin, suggesting a large change in the higher-order structure associated with dissolution and subsequent regeneration of chitin. Since chitin nanoparticles interact with each other less than chitin nanofibers produced by mechanical treatment, the viscosity of nanoparticles was smaller than that of nanofibers. Therefore, it can be prepared at a high concentration. In addition, the chitin nanoparticles can be easily redispersed in water after being concentrated by centrifugation.

Hierarchical surface wrinkles and bumps generated on chitosan films having double-skin layers comprising topmost carrageenan layers and polyion complex layers.

Surface wrinkling to fabricate hierarchical surface topographies has attracted much attention because of the potential and multifunctional applications of hierarchical surface wrinkles beyond uniform wrinkles. Although many reports have described the preparation of hierarchical wrinkles induced by mechanical stress and heat, fabrication through drying-induced shrinkage has hardly been reported. Here we introduce hierarchical surface wrinkles and bumps generated on a chitosan film via the preparation of double-skin layers with κ- and ι-carrageenans, respectively, and subsequent drying. Double-skin layers are fabricated on a swollen chitosan film, called a chitosan hydrogel film, that is soaked first in κ- or ι-carrageenan solution and then in water to remove excess adsorbed κ- or ι-carrageenan. After the film is dried, hierarchical microscopic surface architectures are observed. In the case of the κ-carrageenan system, the wrinkles are hierarchical, consisting of wrinkles (6.2 ± 2.8 µm) that have smaller buckles (0.23 ± 0.09 µm). We reveal that the wrinkles or the smaller buckles are caused by plane inhomogeneous shrinkage between the κ-carrageenan layer and the chitosan film or by the aggregation of the κ-carrageenan layer upon drying, respectively. Interestingly, the ι-carrageenan system showed hierarchical bumps consisting of semispherical bumps (5.6 ± 2.1 µm) that have smaller bumps (0.78 ± 0.27 µm). We reveal that the larger bumps are generated during the immersion of the chitosan hydrogel film into ι-carrageenan solution. The smaller bumps are generated by the aggregation of the ι-carrageenan layer that occurs during drying; this process requires the plane compression strain caused by the shrinkage of the chitosan hydrogel film.

Disease resistance and growth promotion activities of chitin/cellulose nanofiber from spent mushroom substrate to plant.

Some studies have reported the method for treating the spent mushroom substrate (SMS). However, the effective use as a functional raw material based on properties of SMS remains a formidable challenge. In this study, we investigated the usefulness of SMS in agriculture to develop a new method for treating and utilizing it. First, we attempted to isolate chitin/cellulose nanofiber complex (CCNFC) from SMS using chemical pretreatment and mechanical fibrillation. The characterization results like SEM, FT-IR, and XRD showed that we successfully isolated the CCNFC from SMS. Second, we explored the biological activities of the CCNFC for its potential application as a functional agricultural nanomaterial. CCNFC water dispersion with low concentration (0.1 and 1 mg/mL) exhibited significant plant disease resistance and plant growth promotion activities. Our results suggested that SMS may provide a useful source of functional agricultural nanomaterial, which may contribute to treating and applying it in agriculture.

Optimum Preparation Conditions for Highly Individualized Chitin Nanofibers Using Ultrasonic Generator.

α-Chitin derived from crab shells was treated with 30% sodium hydroxide to prepare partially deacetylated chitin with a deacetylation degree of 36%. Partially deacetylated chitin nanofibers were prepared by applying weak ultrasonic energy generated by a domestic ultrasonic cleaner. The deacetylated chitin was easily disintegrated into nanofibers with the aid of electrostatic repulsion and osmotic pressure effect of amino cations on the chitin surfaces. The nanofibers were characterized in terms of yield, morphology, crystallinity, viscosity, and dispersibility. After a series of characterizations, ultrasonication with 45 kHz frequency and 20 min treatment was found to be the optimum conditions for obtaining fine nanofibers with a high yield.

Unique Photophysical Properties of 1,8-Naphthalimide Derivatives: Generation of Semi-stable Radical Anion Species by Photo-Induced Electron Transfer from a Carboxy Group.

The development of anion sensors for selective detection of a specific anion is a crucial research topic. We previously reported a selective photo-induced colorimetric reaction of 1-methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium (MNEI) having a cationic receptor in the presence of molecules having multiple carboxy groups, such as succinate, citrate, and polyacrylate. However, the mechanism underlying this reaction was not clarified. Here, we investigate the photo-induced colorimetric reaction of N-[2-(trimethylammonium)ethyl]-1,8-naphthalimide (TENI), which has a different cationic receptor from MNEI and undergoes the photo-induced colorimetric reaction, and its analogues to clarify the reaction mechanism. The TENI analogues having substituents on the naphthalene ring provide important evidence, suggesting that the colorimetric chemical species were radical anions generated via photo-induced electron transfer from carboxylate to the naphthalimide derivative. The generation of the naphthalimide-based radical anion is verified by 1H NMR and cyclic voltammetry analyses, and photo-reduction of methylene blue is mediated by TENI. In addition, the role of the cationic receptor for the photo-induced colorimetric reaction is investigated with TENI analogues having different hydrophilic groups instead of the trimethylammonium group. Interestingly, the photo-induced colorimetric reaction is observed in a nonionic analogue having a polyethylene glycol group, indicating that the colorimetric reaction does not require a cationic receptor. On the other hand, we reveal that the trimethylammonium group stabilizes the radical anion species. These generation and stabilization phenomena of naphthalimide-based radical anion species will contribute to the development of sophisticated detection systems specific for carboxylate.

Guanidinylation of Chitooligosaccharides Involving Internal Cyclization of the Guanidino Group on the Reducing End and Effect of Guanidinylation on Protein Binding Ability.

In order to synthesize a promising material for developing a novel peptide/protein delivery system, guanidinylation of chitooligosaccharides with 1-amidinopyrazole hydrochloride was investigated herein. The production of guanidinylated chitooligosaccharides was demonstrated by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and elemental analyses. Interestingly, we found that the reducing end in the guanidinylated chitooligosaccharides was converted to a cyclic guanidine structure (2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose structure). This reaction was carefully proven by the guanidinylation of d-glucosamine. Although this is not the first report on the synthesis of the 2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose, it has provided a rational synthetic route using the high reactivity of the reducing end. Furthermore, we found that the interaction between chitooligosaccharides and bovine serum albumin is weak when in a neutral pH environment; however, it is significantly improved by guanidinylation. The guanidinylated chitooligosaccharides are useful not only for the development of a novel drug delivery system but also as a chitinase/chitosanase inhibitor and an antibacterial agent.

Hair growth-promoting activities of chitosan and surface-deacetylated chitin nanofibers.

In this study, the effects of chitosan and surface-deacetylated chitin nanofibrils (SDACNFs) on hair growth were evaluated. In human follicle dermal papilla cells in vitro, chitosan and SDACNFs were shown to increase cell growth on day 3 after the initiation of treatment, together with an increase in the production of fibroblast growth factor-7 (FGF-7) by these cells on day 3. Furthermore, in an in vivo study in mice, chitosan and SDACNF application promoted hair growth. The number of anagen follicles significantly increased compared with that in the control group, whereas the number of telogen follicles significantly decreased in the chitosan and SDACNF groups. In the chitosan and SDACNFs groups, moreover, the expression levels of FGF-7 and Sonic hedgehog were significantly upregulated in hair follicles. Overall, our results demonstrated that chitosan and SDACNFs promoted hair growth and therefore may have applications as novel therapeutic agents for the treatment of hair loss in patients.

Guanidinylated chitosan inspired by arginine-rich cell-penetrating peptides.

We introduce a simple method for producing guanidinylation of chitosan (CS) with 1­amidinopyrazole hydrochloride (AP). Production of GCS is proved via NMR, IR, and elemental analyses. In a reaction using 4.5 eq of AP for 7 days at room temperature, we obtained GCS with 42.3% of degree of guanidinylation (DG). The DG value can be controlled by the reaction time and AP amount. Furthermore, remarkable enhancement of the interaction between GCS and bovine serum albumin by guanidinylation was observed. This simple guanidinylation method for CS could provide novel additives for protein/peptide delivery systems.

Theoretical study on photo-induced processes of 1-methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium halide species: an application of constrained density functional theory.

1-Methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium (MNEI) has potential as a versatile sensor that can measure the electronegativity of anions based on the fluorescence intensity upon irradiation. To clarify the factors that determine the fluorescence intensity, constrained density functional theory (CDFT) was applied to explore the electron transfer (ET) states of MNEI halide species (MNEI-X; X = F, Cl, Br, I). According to the CDFT potential energy surface, intra-molecular ET (SM1) states on MNEI are responsible for the intensity of absorption and fluorescence spectra. However, inter-molecular ET (SET) states between MNEI and X are certainly responsible for fluorescence quenching. Hence, the energetic difference between the SM1 state and the SET state (ΔEM1_ET) is a crucial factor that determines the fluorescence intensity in the spectra of MNEI-X complexes. ΔEM1_ET decreases as the electronegativity of X decreases (i.e., F > Cl > Br > I). This explains the fluorescence intensity of MNEI-X.

Wood-mimetic skins prepared using horseradish peroxidase catalysis to induce surface wrinkling of chitosan film upon drying.

We previously developed bio-based wrinkled surfaces induced by wood-mimetic skins upon drying in which microscopic wrinkles were fabricated on a chitosan (CS) film by immersing it in a phenolic acid solution, followed by horseradish peroxidase (HRP)-catalyzed surface reaction and drying. However, the detailed structure of the resulting wood-mimetic skins, including crosslinking mode and thickness, has not been clarified due to the difficulty of the analysis. Here, we prepare wrinkled films using ferulic acid (FE), vanillic acid (VA), and homovanillic acid (HO) and characterize their structures to clarify the unknown characteristics of wood-mimetic skin. Chemical and structural analyses of wood-mimetic skins prepared using VA and HO indicate that the crosslinking structure in the skin is composed of ionic bonds between CS and an oligophenolic residue generated by the HRP-catalyzed reaction on the CS surface. Moreover, the quantity of these ionic bonds is related to the skin hardness and wrinkle size. Finally, SEM and TOF-SIMS analyses indicate that the skin thickness is on the submicron order (<200nm).

Preparation and biocompatibility of a chitin nanofiber/gelatin composite film.

The development of chitin-based materials with favorable mechanical properties and biocompatibility is an important research goal owing to the wide-ranging practical applications. In this study, a composite film was prepared using chitin nanofibers and gelatin. The CNF/gelatin composite film was highly viscous and had a fine nanofiber structure. The transmittances indicated high transparency, regardless of nanofiber content. The water content of the CNF/gelatin composite film increased linearly as the gelatin content increased. Although the CNF/gelatin composite film did not induce severe inflammation, it strongly induced fibroblast proliferation, indicating high biocompatibility. Based on these results, the films are suitable for biological applications, e.g., tissue engineering, medicines, and cosmetics.

Oral Administration of Surface-Deacetylated Chitin Nanofibers and Chitosan Inhibit 5-Fluorouracil-Induced Intestinal Mucositis in Mice.

This study investigated the prophylactic effects of orally administered surface-deacetylated chitin nanofibers (SDACNFs) and chitosan against 5-fluorouracil (5-FU)-induced intestinal mucositis, which is a common side effect of 5-FU chemotherapy. SDACNFs and chitosan abolished histological abnormalities associated with intestinal mucositis and suppressed hypoproliferation and apoptosis of intestinal crypt cells. These results indicate that SDACNF and chitosan are useful agents for preventing mucositis induced by anti-cancer drugs.

Biobased Wrinkled Surfaces Induced by Wood Mimetic Skins upon Drying: Effect of Mechanical Properties on Wrinkle Morphology.

We previously developed biobased wrinkled surfaces based on wood mimetic skins in which microscopic wrinkles were fabricated on a chitosan film by immersion in a phenolic acid solution, horseradish peroxidase-catalyzed surface reaction, and drying. Here, we prepared a diverse range of wrinkled films by immersion treatment at 30, 40, 50, and 60 °C in p-coumaric acid and then investigated the correlation between wrinkle morphology and mechanical properties. Wrinkle wavelengths gradually decreased as the immersion temperature increased as well as the previous report. In order to clarify the mechanisms responsible for the different wrinkle morphologies, the films were subjected to elastic moduli measurement and GPC analysis after immersion treatment. These experiments provided evidence that the chitosan around the film surface decomposed along with the immersion process. The decomposition was accelerated by higher immersion temperature, suggesting that higher temperatures led to the formation of softer skins, inducing smaller wrinkles. In fact, wrinkle morphologies with this system were predominately determined by the hardness of the wood mimetic skins. This phenomenon is consistent with the fundamentals of surface wrinkling in nature. This study is the first to demonstrate that artificial wrinkling triggered by water evaporation can be controlled by precise control of the surface hardness of soft material.

Dependence of Intestinal Absorption Profile of Insulin on Carrier Morphology Composed of ß-Cyclodextrin-Grafted Chitosan.

The effect of carrier morphology on the intestinal absorption of insulin was investigated using a morphology-tunable polymeric carrier, ß-cyclodextrin-grafted chitosan (BCC). The insulin-BCC complexes were prepared in either acetate or citrate buffer solutions, followed by dilution with phosphate buffer for the administration. The complex had a molecular network structure in the acetate buffer, whereas nanoparticles formed in the citrate buffer. The network structure in the acetate buffer was maintained even after dilution with a phosphate buffer, but the nanoparticles in the citrate buffer caused aggregation after dilution. Both complexes enhanced the intestinal absorption of insulin. Interestingly, their absorption profiles were totally different; prompt absorption was observed for the complex prepared in acetate buffer, whereas sustained absorption was observed for the complex prepared in citrate buffer. The difference in the absorption patterns was attributed to the difference in the complex morphology. Next, penetratin, a cell-penetrating peptide, was grafted to BCC to find further improvement in the absorption behavior. A simple mixture of penetratin and BCC was also effective. An oral administration study was also conducted in mice to observe effective suppression of glucose levels, which was further enhanced by coadministration of penetratin. Thus, BCC was proven to be an effective carrier for enhancing oral absorption of peptide drugs, and it is suggested that the carrier morphology is also an important factor that influences the absorption profile.

Protein/CaCO3/Chitin Nanofiber Complex Prepared from Crab Shells by Simple Mechanical Treatment and Its Effect on Plant Growth.

A protein/CaCO3/chitin nanofiber complex was prepared from crab shells by a simple mechanical treatment with a high-pressure water-jet (HPWJ) system. The preparation process did not involve chemical treatments, such as removal of protein and calcium carbonate with sodium hydroxide and hydrochloric acid, respectively. Thus, it was economically and environmentally friendly. The nanofibers obtained had uniform width and dispersed homogeneously in water. Nanofibers were characterized in morphology, transparency, and viscosity. Results indicated that the shell was mostly disintegrated into nanofibers at above five cycles of the HPWJ system. The chemical structure of the nanofiber was maintained even after extensive mechanical treatments. Subsequently, the nanofiber complex was found to improve the growth of tomatoes in a hydroponics system, suggesting the mechanical treatments efficiently released minerals into the system. The homogeneous dispersion of the nanofiber complex enabled easier application as a fertilizer compared to the crab shell flakes.

Preparation of chitin nanofibers by surface esterification of chitin with maleic anhydride and mechanical treatment.

Esterification with maleic anhydride significantly improved the mechanical disintegration of chitin into uniform 10-nm nanofibers. Nanofibers with 0.25° of esterification were homogeneously dispersed in basic water due to the carboxylate salt on the surface. Esterification proceeded on the surface and did not affect the relative crystallinity. A cast film of the esterified chitin nanofibers was highly transparent, since the film was free from light scattering.

Intermolecular electron transfer states of 1-methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium iodide obtained by constrained density functional theory.

Electron transfer (ET) states of 1-methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium iodide are responsible for its photophysics. Investigation of an ET state based on constrained density functional theory (CDFT) revealed that nonradiative decay from the ET excited state is mediated by the interaction of the iodine atom with the 1,8-naphthalimide or the imidazolium group.

Chitin nanofibrils suppress skin inflammation in atopic dermatitis-like skin lesions in NC/Nga mice.

We evaluated the effect of chitin nanofibril (CNF) application via skin swabs on an experimental atopic dermatitis (AD) model. AD scores were lower, and hypertrophy and hyperkeratosis of the epidermis were suppressed after CNF treatment. Furthermore, inflammatory cell infiltration in both the epidermis and dermis was inhibited. CNFs also attenuated histological scores. The suppressive effects of CNFs were equal to those of corticosteroid application; however, chitin did not show these effects. CNF application might have anti-infllammatory effects via suppression of the activation of nuclear factor-kappa B, cyclooxygenase-2, and inducible nitric oxide synthase. In an early-stage model of experimental AD, CNFs suppressed AD progression to the same extent as corticosteroids. They also suppressed skin inflammation and IgE serum levels. Our findings indicate that CNF application could aid in the prevention or treatment of AD skin lesions.

Biomineralization of calcium phosphate crystals on chitin nanofiber hydrogel for bone regeneration material.

We previously reported a chitin nanofiber hydrogel from squid pen ß-chitin by a simple NaOH treatment. In the present study, a calcium phosphate/chitin nanofiber hydrogel was prepared for bone tissue engineering. Calcium phosphate was mineralized on the hydrogel by incubation in a solution of diammonium hydrogen phosphate solution followed by calcium nitrate tetrahydrate. X-ray diffractometry and Fourier transform infrared spectroscopy showed the formation of calcium phosphate crystals. The morphology of the calcium phosphate crystals changed depending on the calcification time. After mineralization, the mechanical properties of the hydrogel improved due to the reinforcement effect of calcium phosphate crystal. In an animal experiment, calcium phosphate/chitin nanofiber hydrogel accelerated mineralization in subcutaneous tissues. Morphological osteoblasts were observed.