Temperature-Responsive Polysaccharide Microparticles Containing Nanoparticles: Release of Multiple Cationic/Anionic Compounds.

Most drug carriers used in pulmonary administration are microparticles with diameters over 1 µm. Only a few examples involving nanoparticles have been reported because such small particles are readily exhaled. Consequently, the development of microparticles capable of encapsulating nanoparticles and a wide range of compounds for pulmonary drug-delivery applications is an important objective. In this study, we investigated the development of polysaccharide microparticles containing nanoparticles for the temperature-responsive and two-step release of inclusions. The prepared microparticles containing nanoparticles can release two differently charged compounds in a stepwise manner. The particles have two different drug release pathways: one is the release of nanoparticle inclusions from the nanoparticles and the other is the release of microparticle inclusions during microparticle collapse. The nanoparticles can be efficiently delivered deep into the lungs and a wide range of compounds are released in a charge-independent manner, owing to the suitable roughness of the microparticle surface. These polysaccharide microparticles containing nanoparticles are expected to be used as temperature-responsive drug carriers, not only for pulmonary administration but also for various administration routes, including transpulmonary, intramuscular, and transdermal routes, that can release multiple drugs in a controlled manner.

Precise Control of the Surface and Internal Morphologies of Porous Particles Prepared Using a Spontaneous Emulsification Method.

Porous particles with controllable surface and internal morphologies were successfully prepared by a "one-step mechanical emulsification" technique via the control of spontaneous emulsification where self-emulsification is followed by mechanical emulsification. The morphological changes in the porous particles were determined not by the preparation conditions of the water-in-oil-in-water (w/o/w) emulsion but by the proportion of solvents that favors the stabilization of the spontaneously prepared water-in-oil (w/o) emulsion droplets acting as porogens. The proposed method for controlling the morphology of the porous particles could be applied to all particle-preparation systems based on emulsion-solvent evaporation using organic solvents. The methodology for the morphological control of porous particles independent of the concentration or composition of the polymer is considered valuable for future investigations into the aerodynamic performance and drug-release behavior of biomedical porous particles with complex shapes.

A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures.

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.

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.

PLA- and PLA/PLGA-Emulsion Composite Biomaterial Sheets for the Controllable Sustained Release of Hydrophilic Compounds.

In the present study, by spin-coating a solution containing w/o (water-in-oil) emulsions and hydrophobic polymers, we obtained sheets possessing uniformly dispersed w/o emulsions. We performed release experiments for more than 100 days and clarified the effects of the number of layers, the sheet-forming polymers (polylactide (PLA), poly(lactic-co-glycolic acid (PLGA)), the ratio of organic solvent to water, and the composition of block copolymers on the release properties of the sheets. For a variety of sheets, we successfully achieved the sustained release of compounds from the sheets for 100⁻150 days. The sustained-release of compounds occurred because the compounds had to diffuse into polymer networks after their release from the emulsions. Interestingly, we observed an inflection point in the release profiles at around 50 days; that is, the sheet exhibited a "two-step" release behavior. The results obtained in the present study provide strong evidence for the future possibility of the time-programmed release of multiple compounds from sheets.

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.

Synergic modulation of the inflammatory state of macrophages utilizing anti-oxidant and phosphatidylserine-containing polymer-lipid hybrid nanoparticles.

Inflammatory activation of macrophages is a key factor in chronic inflammatory diseases such as ulcerative colitis. The excessive production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) by macrophages causes oxidative stress during the inflammatory response and exaggerates inflammatory lesions in ulcerative colitis. Inhibition of the inflammatory activation of macrophages is a promising treatment for chronic inflammatory diseases. Here, we prepared self-filling polymer-lipid hybrid nanoparticles (PST-PLNPs) consisting of poly dl-lactic acid as a hydrophobic biodegradable polymer core encapsulating α-tocopherol (T) and phosphatidylserine (PS) both on the surface and interior of the particle. We confirmed the anti-inflammatory response of these hybrid nanoparticles in activated murine macrophages. PS has anti-inflammatory effects on macrophages by modulating the macrophage phenotype, while α-tocopherol is an antioxidant that neutralizes ROS. We found that PS-containing (PS-PLNPs) and PS plus α-tocopherol-containing (PST-PLNPs) polymer-lipid hybrid nanoparticles significantly increased the viability of lipopolysaccharide (LPS)-treated macrophages compared with phosphatidylcholine-containing PLNPs. PST-PLNPs had a better effect than PS-PLNPs, which was attributed to the synergy between PS and α-tocopherol. This synergic action of PST-PLNPs reduced NO and pro-inflammatory cytokine (IL-6) production and increased anti-inflammatory cytokine (TGF-ß1) production when incubated with activated macrophages. Thus, these self-filling biodegradable polymer-lipid hybrid nanoparticles (PST-PLNPs) containing anti-oxidant and anti-inflammatory molecules might be potential alternative drug carriers to liposomes and polymeric nanoparticles for the treatment of chronic inflammatory diseases such as ulcerative colitis.

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.

Clinical subsets associated with different anti-aminoacyl transfer RNA synthetase antibodies and their association with coexisting anti-Ro52.

OBJECTIVE: To characterize clinical features of polymyositis/dermatomyositis (PM/DM) patients with different anti-aminoacyl transfer RNA synthetase (ARS) antibodies and their association with anti-Ro52. METHODS: Autoantibodies in sera from 97 Japanese patients (36 PM, 56 DM, and 5 clinically amyopathic DM), who satisfied Bohan and Peter or modified Sontheimer's criteria, were characterized by immunoprecipitation and enzyme-linked immunosorbent assay. Clinical information was from medical records. Features associated with different anti-ARS and anti-Ro52 antibodies were analyzed. RESULTS: The prevalence of anti-ARS was similar to other studies (Jo-1, 22%; EJ, 4%; OJ, 1%; PL-12, 1%), except for a high prevalence of anti-PL-7 (12%), which allowed us to characterize patients carrying this specificity. Serum creatine kinase >3000 IU/l was less common in anti-PL-7-positive patients (57%) than anti-Jo-1-positive patients (18%) (p = 0.0328) and was not found in anti-EJ-positive individuals. Interstitial lung disease was common in anti-ARS-positive patients (97%) (p < 0.0001 vs. 48% in anti-ARS-negative). Anti-Ro52 antibodies were frequently detected with anti-ARS (59%) (57% in anti-Jo-1, 67% in anti-PL-7) (vs. 21% in anti-ARS-negative, p < 0.0002). Anti-Ro52 was associated with overlap syndrome (26%) (vs. 7% in anti-Ro52-negative, p = 0.0119). CONCLUSIONS: Patients with different anti-ARS in combination with anti-Ro52 appear to be associated with distinctive clinical subsets.

Poly(ɛ-caprolactone) (PCL)-polymeric micelle hybrid sheets for the incorporation and release of hydrophilic proteins.

Sheets have several advantages over conventional gel- or particle-type drug carriers. Sheets have several notable attributes: sheets' size and shape are easily adjustable, sheets are highly accessible in surgery, and sheets have a large contact area relative to drug-targeting sites. However, it is difficult to incorporate hydrophilic proteins into hydrophobic sheets and to release the proteins over the long term in a sustained manner. In the present study, we show that "poly(ɛ-caprolactone) (PCL)-polymeric micelle hybrid sheets" can be used for the incorporation and release of hydrophilic proteins. Polymeric micelles (i.e., spaces that can incorporate hydrophilic compounds) are, in this study, uniformly dispersed in hydrophobic and biocompatible biomaterial sheet. We have clarified that the composition of block copolymer, methoxy-terminated poly(ethylene glycol)-block-poly(ɛ-caprolactone) (CH3O-PEG-b-PCL), can affect two variables: the stability of w/o emulsion and the release properties of the resulting sheets, by means of visual qualitative observations, newly developed quantitative analyses (advanced fractal analysis, advanced FD) based on deviation of the fractal dimension (FD), and release experiments. We clarified that the release behavior of BSA was affected by the composition of the block copolymers and the resulting emulsion. The results obtained in this paper show that the hydrophobic sheets in which polymeric micelles providing hydrophilic spaces were dispersed could be an effective platform for incorporating and releasing hydrophilic proteins.

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.

[A patient with Creutzfeldt-Jakob disease supported by home medical care from the stage of disease progression to death through hospital-clinic cooperation and medical-welfare cooperation].

The patient was a 63-year-old woman who presented with slowness of speech after cerebral infarction. Diffusion-weighted MR images and investigations of cerebrospinal fluid showed abnormal values, and the patient was diagnosed as having sporadic Creutzfeldt-Jakob disease(CJD). This is an intractable disease and affects one in one million people; it progresses relatively rapidly, eventually resulting in death. For procedures such as intravenous fluid replacement and the treatment of pressure sores, we require thorough hand washing, eye protection, and disposal of gloves and dressings by incineration. It is desirable for patients to spend the limited amount of time available to them peacefully at home with their family. Visiting physicians and nurses need to take the initiative in sharing information obtained from the CJD infection control guidelines and core hospitals with welfare personnel such as caregivers, in order to provide correct information on all aspects of patient care and the management of this disease in the home environment. Excellent supportive care was provided for the patient at home, and she passed away with her family by her side.

Thermoresponsive synthetic polymer-microtubule hybrids.

Thermoresponsive hybrids consisting of synthetic polymers and microtubules (MTs), i.e., assemblies of tubulins, were prepared by bonding MTs covalently to a few reactive units in a macromolecular strand. The hybrids exhibited the gel/sol transition because of the "assembling of tubulins to MTs/disintegrating of MTs to tubulins" by the temperature change between 37 and 4 °C, respectively. The viscoelastic behaviors of the hybrid gels depended upon the quantity of polymer feed and the amount of resulting covalent bonds between the polymers and tubulin units. Furthermore, in a confined space of a thin and long rectangular cell with the temperature gradient from 4 °C (cold terminal) to 37 °C (warm terminal), the sol state hybrid turned to the gel state that propagated from the warm terminal toward the cold terminal to form uniaxially oriented MT arrays. Upon changing the temperature of the whole system between 37 and 4 °C, the uniaxial arrays appeared/disappeared reversibly.

Design of novel sheet-shaped chitosan hydrogel for wound healing: a hybrid biomaterial consisting of both PEG-grafted chitosan and crosslinkable polymeric micelles acting as drug containers.

In this study, we successfully prepared a novel "sheet-shaped" chitosan hydrogel for wound healing consisting of both PEG-g-chitosan and a crosslinkable polymeric micelle. The study's findings clarify that the PEG modification percentage (PMP) of PEG-g-chitosan increased proportionally as the weight ratio of PEG/chitosan increased. Furthermore, the positive second virial coefficient of PEG-g-chitosans from a Debye plot strongly suggests that the PEG modification greatly improved the solubility of the water-insoluble chitosan. Finally, the "sheet-shaped" "flexible" hydrogel formed by mixing solutions containing either PEG-g-chitosan with moderate PMP or polymeric micelles exhibited the highest storage modulus. The sheet itself exhibited an attractive feature insofar as polymeric micelles, which can act as drug containers facilitating the incorporation and the gradual release of drugs, are covalently immobilized in the polymeric network of the hydrogel. The results obtained in the present study show that the hybrid PEG-g-chitosan hydrogel containing crosslinkable polymeric micelles has the potential to address the need for novel functional biomaterials.