Solubilized eggshell membrane supplies a type III collagen-rich elastic dermal papilla.

Signs of aging in facial skin correlate with lifespan and chronic disease; however, the health of aging skin has not been extensively studied. In healthy young skin, the dermis forms a type III collagen-rich dermal papilla, where capillary vessels supply oxygen and nutrients to basal epidermal cells. Chicken eggshell membranes (ESMs) have been used as traditional medicines to promote skin wound healing in Asian countries for many years. Previously, we designed an experimental system in which human dermal fibroblasts (HDFs) were cultured on a dish with a solubilized ESM (S-ESM) bound to an artificial phosphorylcholine polymer; we found that genes that promoted the health of the papillary dermis, such as those encoding type III collagen, were induced in the S-ESM environment. The present study found that a gel with a ratio of 20% type III/80% type I collagen, similar to that of the baby skin, resulted in a higher elasticity than 100% type I collagen (p < 0.05) and that HDFs in the gel showed high mitochondrial activity. Thus, we decided to perform further evaluations to identify the effects of S-ESM on gene expression in the skin of hairless mice and found a significant increase of type III collagen in S-ESM. Picrosirius Red staining showed that type III collagen significantly increased in the papillary dermis after S-ESM treatment. Moreover, S-ESM application significantly improved human arm elasticity and reduced facial wrinkles. ESMs may have applications in extending lifespan by reducing the loss of tissue elasticity through the increase of type III collagen.

Chitosan gel sheet containing drug carriers with controllable drug-release properties.

We prepared the "sheet-type hydrogel" (gel sheet), a sheet consisting of PEG-grafted chitosan and cross-linkable polymeric micelles, that were expected to be used for wound healing. We optimized the PEG-modification process, evaluated the strain-dependence of the gel's properties to obtain flexible gel sheets, and evaluated the drug-release properties of the gel sheets. Finally, we succeeded in observing that the release of the antibiotic tetracycline (TET) from the gel sheet in which TET existed only in the cross-linkers was lower than the release of TET from the sheet in which TET was dispersed in the polymer networks. Our research demonstrates that the strategy of incorporating drug carriers into gel sheets might benefit the construction of biomaterials with controllable drug-release properties.

Chitosan Gel Sheet Containing Polymeric Micelles: Synthesis and Gelation Properties of PEG-Grafted Chitosan.

Wound-dressing sheet biomaterials can cover wound sites and enhance wound healing. In this study, a detailed evaluation of the factors affecting both the PEG modification percentage (PMP) in poly(ethylene glycol) (PEG)-grafted chitosan synthesis and the gelation properties of PEG-grafted chitosan was presented for constructing our novel hybrid hydrogel sheet consisting of PEG-grafted chitosan (a gel-forming polymer) and a reactive polymeric micelle (a crosslinker). It was confirmed that various factors (i.e., the weight ratio of PEG/chitosan, the pH of the buffer solution, reaction times, and reaction temperatures) in the preparation stage of PEG-grafted chitosans affected the PMP of PEG-grafted chitosans. Furthermore, the PMP of PEG-grafted chitosans affected their gelation properties. Finally, a 'flexible' hydrogel sheet that can be reversibly dried and moistened was successfully obtained. The dried rigid, thin sheet is expected to be suitable for stable preservation. The results obtained in this paper show that the incorporation of drug carriers into biomaterials is a novel approach to improve functionality.

Porous PLGA microparticles formed by "one-step" emulsification for pulmonary drug delivery: The surface morphology and the aerodynamic properties.

In the present study, by using a newly developed one-step emulsification technique, we tried to prepare porous PLGA particles having a proper diameter and surface morphology in order to achieve both a high efficient delivery of the particles to the lungs and a phagocytosis-avoidance ability. We found that our porous particles have the very low tapped density of 0.04g/cm3. Experimental and theoretical studies strongly suggest that the shape factor should not be determined only by the outline of the particles, although previous research assigned a value of 1 to the shape factor for particles regardless of the presence of pores and their distribution. We found the possibility that our porous particles both had specific internal structures induced by spontaneous emulsification and exhibited unusual aerodynamic performance.

Successful PEGylation of hollow encapsulin nanoparticles from Rhodococcus erythropolis N771 without affecting their disassembly and reassembly properties.

We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent. The nanoparticle obtained in the present study has the potential to incorporate hydrophilic compounds in the internal cavity of the particle by reversibly controllable disassembly and reassembly. The hollow PEGylated encapsulin nanoparticle can be used as a drug carrier for the delivery of hydrophilic biopolymers in future medical applications.

Dual drug release from hydrogels covalently containing polymeric micelles that possess different drug release properties.

In the present study, we designed hydrogels for dual drug release: the hydrogels that covalently contained the polymeric micelles that possess different drug release properties. The hydrogels that were formed from polymeric micelles possessing a tightly packed (i.e., well-entangled) inner core exhibited a higher storage modulus than the hydrogels that were formed from the polymeric micelles possessing a loosely packed structure. Furthermore, we conducted release experiments and fluorescent observations to evaluate the profiles depicting the release of two compounds, rhodamine B and auramine O, from either polymeric micelles or hydrogels. According to our results, (1) hydrogels that covalently contains polymeric micelles that possess different drug release properties successfully exhibit the independent release behaviors of the two compounds and (2) fluorescence microscopy can greatly facilitate efforts to evaluate drug release properties of materials.

Design of Dithiobenzoate RAFT Agent Bearing Hydroxyl Groups and Its Application in RAFT Polymerization for Telechelic Diol Polymers.

RAFT polymerization is attractive for its reliability, facile operation, and high tolerance to a wide variety of monomers, functional groups, solvents, and temperatures. Herein, we report the RAFT-based synthesis of well-defined polymers bearing hydroxyl groups at two terminals by using various monomers. We found that the molecular weight of obtained polymers was half that of a target value when a trithiocarbonate-type chain transfer agent (CTA) was used, suggesting that the polymers unexpectedly cleaved at the middle of the polymer chain as the reaction was proceeding. To address the problem, we synthesized a novel "dithiobenzoate"-type CTA, 2-[N-(2-hydroxyethyl)carbamoyl]prop-2-yl 4-hydroxydithiobenzoate (HECPHD), which bears hydroxyl groups at both terminals, and we succeeded in RAFT polymerization with various monomers without a cleavage of the polymers.

Poly(ε-caprolactone) (PCL) hybrid sheets containing polymeric micelles: Effects of inner structures on the material properties of the sheets.

In the present paper, we clarify the effects that the composition of three types of sheets-the PCL sheet, the PCL-BC (PCL-block copolymer composite) sheet, and the PCL-PM (PCL-polymeric micelle composite) sheet-can have on (1) the sheets' inner structure, (2) the dispersity of hydrophilic compounds in the sheets, (3) the sheets' mechanical properties, and (4) the sheets' degradability. Our results show that (1) the PCL-PM sheet can disperse hydrophilic compounds uniformly, (2) the molecular state (free or micellar) of a co-existing compound (PEG-b-PCL block copolymers) affects the strength and the inner structures of the sheets, whereas the presence of a co-existing compound affects the flexibility of the sheets, and (3) according to our degradation experiment, hard-to-handle PCL having extremely low hydrolysis could serve as materials with a controllable surface morphology by the effective use of co-existing compounds. The results obtained in this paper show that the PCL-CM sheet, with its uniformly dispersed polymeric micelles providing hydrophilic spaces, could be an effective biomaterial platform for incorporating hydrophilic polymers.

Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771.

The encapsulin nanocompartment from Rhodococcus erythropolis N771 (Reencapsulin) was expressed and purified in wild-type and C-terminally His-tagged forms. Negative-stained transmission electron microscopy, field-flow fractionation combined with multi-angle light scattering and dynamic light scattering analyses showed that 60 Reencapsulin monomers were assembled as a spherical particle with a diameter of 28 nm. Heterogeneous guest proteins such as EGFP and firefly luciferase were packaged into the internal cavity of the Reencapsulin nanocompartment by fusing the C-terminal 37-amino-acid sequence of the R. erythropolis N771 DypB peroxidase to the C-terminus. Reencapsulin has the potential to package target proteins in its internal cavity and/or display them on its external surface, making it a feasible carrier for nanotechnology applications.

Unexpected and successful "one-step" formation of porous polymeric particles only by mixing organic solvent and water under "low-energy-input" conditions.

We found that porous particles were unexpectedly obtained in a "one-step" manner only by mixing an organic solvent and water under "low-energy-input" (i.e., low-homogenization-rate) conditions. This phenomenon was attributable to the unexpected formation of the spontaneously formed water-in-oil (w/o) emulsions in the droplets of o/w emulsions. The unexpected formation resulted in the successful formation of water-in-oil-in-water (w/o/w) emulsions instead of o/w emulsions, although the mixed solution containing both an organic solvent and water were simply emulsified in the presence of block copolymers. The present study clarifies the effects of the various preparation conditions on the morphology of unexpected w/o/w emulsions and resulting particles. The porous particles are expected to be suitable drug carriers for pulmonary delivery. The results obtained in the present study show that a newly developed one-step emulsification can be a powerful and facile technique for preparing porous polymeric particles.

Development of PEG-PLA/PLGA microparticles for pulmonary drug delivery prepared by a novel emulsification technique assisted with amphiphilic block copolymers.

We developed a novel "spray dry-based" method for preparing surface-modified particle via "block copolymer-assisted" emulsification/evaporation for pulmonary drug delivery. The method included three steps: (1) o/w emulsion containing both hydrophobic polymers and amphiphilic block copolymers was obtained by emulsification of water and a polymer-containing organic solvent, (2) the o/w emulsion was misted with a nebulizer, and (3) the emulsion mists were dried by a heater. In this way, the hydrophobic polymers and the hydrophobic part of the amphiphilic block copolymers gradually tangled during the evaporation of organic solvents from the o/w emulsion. Consequently, the hydrophilic polymer chain was introduced on the particle surface. The particle surface can be easily modified although there are no reactive groups in the hydrophobic polymer molecules. We successfully obtained dry PEG-PLA/PLGA microparticles by controlling the weight ratio of the block copolymer and the hydrophobic polymer. The introduction of PEG to the particle surface involves an increase in the Zeta potential of the particles. Interestingly, the "dimpled" microparticles having a diameter of approximately 2 µm were obtained. The "dimpled" microparticles can serve as drug carriers for pulmonary drug delivery, because the particles have a large surface area. We expect that this novel surface-modification technique will enable efficient fabrication of particles in drug delivery systems.