Rie Wakabayashi

Enzymatic preparation of a redox-responsive hydrogel for encapsulating and releasing living cells

Horseradish peroxidase-mediated oxidative cross-linking of a thiolated poly(ethylene glycol) is promoted in the absence of exogenous hydrogen peroxide, by adding a small amount of a phenolic compound under physiological conditions. The prepared hydrogel can encapsulate and release living mammalian cells.

Effective transgene expression without toxicity by intraperitoneal administration of PEG-detachable polyplex micelles in mice with peritoneal dissemination

Block copolymer of poly(ethylene glycol)-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-P[Asp(DET)]) has been originally introduced as a promising gene carrier by forming a nanomicelle with plasmid DNA. In this study, the polyplex micelle of PEG-SS-P[Asp(DET)], which disulfide linkage (SS) between PEG and cationic polymer can detach the surrounding PEG chains upon intracellular reduction, was firstly evaluated with respect to in vivo transduction efficiency and toxicity in comparison to that of PEG-P[Asp(DET)] in peritoneally disseminated cancer model. Intraperitoneal (i.p.) administration of PEG-SS-P[Asp(DET)] polyplex micelles showed a higher (P<0.05) transgene expression compared with PEG-P[Asp(DET)] in tumors. In contrast, the delivered distribution of the micelles was not different between the two polyplex micelles. PEG-SS-P[Asp(DET)] micelle encapsulating human tumor necrosis factor α (hTNF-α) gene exhibits a higher antitumor efficacy against disseminated cancer compared with PEG-P[Asp(DET)] or saline control. No hepatic and renal toxicities were observed by the administration of polyplex micelles. In conclusion, PEG-detachable polyplex micelles may represent an advantage in gene transduction in vivo over PEG-undetachable polyplex micelles after i.p. administration for peritoneal dissemination of cancer.

Ionic liquid-mediated transcutaneous protein delivery with solid-in-oil nanodispersions

As a potentially safe and non-invasive vaccination method, transcutaneous immunization represents an attractive alternative to conventional vaccine delivery by injection. However, the development of transcutaneous immunization has remained a challenge for a large number of hydrophilic macromolecules including protein and peptide antigens. We report a novel ionic liquid (IL)-mediated transcutaneous vaccine formulation consisting of a solid-in-oil (S/O) nanodispersion of antigen coated with pharmaceutically accepted surfactants dispersed in IL-containing oil. The introduction of the IL [C12mim][Tf2N] (1-dodecyl-3-methyl imidazolium bis(trifluoromethyl sulfonyl) amide) as a penetration enhancer in the formulation significantly enhanced the skin permeability of ovalbumin (OVA), a model antigen. It was also found that the IL-mediated S/O nanodispersion obtained high levels of OVA-specific serum IgG compared with both S/O nanodispersions without IL and PBS control. These findings clearly indicate that ILs – which are potentially attractive “green” and “designer” solvents – could serve as potential skin penetration enhancers in transcutaneous vaccination for hydrophilic macromolecules.

Enzymatic Fabrication of Protein-Decorated Gold Nanoparticles by the Aid of Artificial Peptides with Gold-Binding Affinity

Here, we report a new approach for the biofabrication of protein-immobilized gold nanoparticles (Au NPs), using oxidoreductase with gold-binding peptide-tagged recombinant proteins. The reduction of Au ions to Au(0) was achieved using a natural electron-donating cofactor, nicotinamide adenine dinucleotide, which was regenerated by the glycerol dehydrogenase (GLD) enzyme. First, we selected the A3 peptide (AYSSGAPPMPPF) as a gold binding moiety. The A3 peptide was introduced to the C-terminus of fusion proteins of immunoglobulin G (IgG)-binding domains of protein G and protein A. In the presence of the recombinant protein, the GLD-catalyzed cofactor reduction resulted in the efficient in situ fabrication of Au NPs immobilized with the fusion protein. Moreover, the protein-immobilized Au NPs were shown to have IgG binding activity. Although the A3 peptide had the ability to stabilize Au NPs, the results suggested that its binding affinity for Au NPs was unexpectedly weaker than that of His-tag. A cysteine residue was thus introduced to a recombinant protein adjacent to the A3 peptide. Finally, an artificial peptide, comprising A3 sequence with the C-terminal single cysteine residue, enabled the stable display of a fusion protein while maintaining its IgG binding activity through the Au-S bond. This enzyme-assisted one-pot methodology for protein-Au NPs conjugation offers one potent route for the facile fabrication of biomolecule-decorated metal NPs.

Unexpected Effects of Terminal Olefins on a Cooperative Recognition System that Implicate Olefin–Olefin Interactions†

To design receptors or building blocks that are useful for constructing supramolecular architectures, multiple noncovalent, relatively weak interactions are essential to realize flexible stimuli-responsive features. The systematic study of such weak but crucial interactions is of great importance in both synthetic and biological systems, but seem to be rather difficult to estimate because of their weakness or lability to the external environment. Among many approaches for understanding such weak interactions, Wilcox and co-workers introduced a smart method for the evaluation of CH–p interactions using a “molecular torsion balance”, in which the rotational barrier between folded (with interaction) and unfolded (without interaction) states is used to calculate the force. Diederich and co-workers applied a chemical doublemutant system to the molecular torsion balance described by Wilcox for the measurement of CF–amide interactions, thus providing evidence that the stability is less than 4 kJmol . In our recent study on the template synthesis of a pseudorotaxane complex facilitated by allosterism, we noticed unexpectedly that the cooperative binding behavior of a host molecule bearing olefin substituents at the periphery of the binding sites are significantly different from those of a non-olefinic counterpart. As the structures are basically the same except for the presence or the absence of the terminal olefins, this difference in cooperativity (see below) seems to arise from the “interaction” among the olefin substituents. Herein, we report the influence of the terminal olefin substituents, which have been introduced into a series of host molecules, on their allosteric behavior. Based on the systematic investigation of the binding properties and the structural analysis of the olefinic host molecules, we have confirmed that the olefinic host molecules elicit a decrease in cooperativity and an increase in affinity for the first guest molecule. These findings clearly indicate that the weak interactions that exist between the olefin substituents can be detected using the allosteric recognition systems. We first employed allosteric host molecules bearing four zincporphyrin units as recognition sites (1a–1c) to demonstrate the effect of terminal olefins on the recognition events (Scheme 1).These molecules have been previously reported to bind diamine molecules (4) with a 1:2 stoichiometry in an allosteric manner. 5] The binding of the first diamine molecule allows the recognition site for the second molecule to be predisposed to binding another diamine molecule because of the restriction of rotation around the butadiyne axis; as a result, 1a–1c exhibit positive homotropic allosterism toward 4 (Figure 1a). For the UV/Vis spectra of 1a, the bathochromic shifts in the Q bands were observed upon successive addition of 4 in CHCl3; [7] these changes arose from the formation of coordination bonds between zincporphyrins in 1 and amino groups in 4. The degree of cooperativity can be analyzed by using the Hill equation: log(y/1 y) = nlog[guest] + log K, where the values for n and K are the Hill coefficient and the association constant, respectively. It is known that a high n value results from the increased cooperativity in a guest-binding process, and the maximum n value is equal to the number of binding sites of a host molecule. We previously reported that the diamine binding to 1a has the Hill coefficient n = 1.9, 7] thus indicating that two guest molecules are bound cooperatively. The n values of 1b and 1c, bearing two and six pairs of terminal olefins, respectively, were slightly smaller than that of 1a ; n = 1.8 for 1b and n = 1.6 for 1c. 7] Although the observed difference in n values is relatively small, the comparison of the first association constants (K1), which was evaluated by a standard non-linear curve-fitting method, reveals the significant difference among them; K1 = 1.6 10m 1 for 1a, K1 = 4.7 10 m 1 for 1 b, and K1 = 8.3 10m 1 for 1c. Interestingly, the n and K1 values in the guest recognition correlate with the number of terminal olefins in the host molecules; that is, as the number of olefinic groups increases, the n value decrease and the K1 value increase. According to the Monod–Wyman–Changeux model for positive homotropic allosterism, a degree of cooperativity (the n value) closely correlates with the L value, where L is defined as [T (an unbound conformation)]/[R (a bound conformation)]. In this model, the higher L value results in a higher n value, which supports the view that one can qualitatively assume the conformation of a host molecule without guest(s) from the n value. Our finding, which shows that the host molecules bearing terminal olefin substituents [*] Dr. R. Wakabayashi, Dr. T. Ikeda, Dr. Y. Kubo, Prof. S. Shinkai Department of Chemistry and Biochemistry Graduate School of Engineering, Kyushu University Fukuoka 819-0395 (Japan) E-mail: seiji_center@mail.cstm.kyushu-u.ac.jp

Supramolecular Assemblies of Polyaniline through Cooperative Bundling by a Palladium‐Complex‐Appended Synthetic Cross‐Linker

A synthetic cross-linker (1) bearing two binding sites for imine moieties in polyaniline has been used to organize polyaniline emeraldine base (EB) or polyaniline emeraldine salt (ES) into aligned assemblies. Complex 1 exhibited highly cooperative binding toward mEB (a repeating unit analogue of EB), for which the association constants K(1) and K(2) were evaluated to be 2.2x10(4) and 9.4x10(5) M(-1), respectively. (1)H NMR studies revealed that the diimine moieties in mEB are recognized cooperatively in anti-conformation by 1. The ordered structures organized from 1 and EB or ES were efficiently formed through supramolecular bundling. These ordered assemblies have a periodicity of 2.5 nm that was confirmed by means of transmittance electron microscopy (TEM) and high-resolution TEM (HRTEM). The cooperative binding would be important for the bundling and alignment of the polymers, as evidenced by the fact that neither reference compounds 2 nor 3 were capable of producing similar assemblies. The electric conductivity of the samples was measured and the results were discussed.

Protein-Grafted Polymers Prepared Through a Site-Specific Conjugation by Microbial Transglutaminase for an Immunosorbent Assay

Protein-polymer conjugates have been developed in many fields. Most hybrids are composed of one protein attached to one or several polymer chains. The other form of hybrid involves the construction of multiple proteins on one polymer chain, thereby facilitating protein assemblies that provide multivalent effects. Unfortunately, synthetic methods for production of these types of hybrids are limited and challenging because precise control of the conjugation sites is needed. Herein, a novel synthetic polymer that can enzymatically assemble multiple proteins was developed. Polyacrylamide grafted with multiple microbial transglutaminase (MTG)-recognizable peptide derivatives was synthesized, and MTG-catalyzed site-specific conjugation of proteins with the polymer was achieved. The application for immunological biosensing was demonstrated using the assembly of a fusion protein composed of antibody-binding and enzyme moieties. This enzymatic method to synthesize a one-dimensional protein assembly on a synthetic polymer is versatile and can be expanded to a wide range of applications.

Split Spy0128 as a Potent Scaffold for Protein Cross-Linking and Immobilization

Site-specific cross-linking techniques between proteins and additional functional groups have become increasingly important for expanding the utility of proteins in biochemistry and biotechnology. In order to explore powerful techniques for practical bioconjugation applications, we have validated a technique mediated by a unique property of Streptcoccus pyogenes pilin subunit Spy0128, an autocatalytic intramolecular isopeptide formation in Spy0128. Recently, it has been revealed that Spy0128 can be split into two fragments (split-Spy0128 (residues 18-299 of Spy0128) and isopeptag (residues 293-308 of Spy0128)) that were capable of forming an intermolecular covalent complex. We focused on this unique reconstitution property and first studied the bioconjugation of blue and green fluorescent proteins, enabling the direct monitoring of cross-linking reactions by Förster resonance energy transfer (FRET). A fluorescence lifetime study shows that spatial control of two proteins on the Spy0128 scaffold is possible when one protein is fused to the N-terminus of split-Spy0128 and another one is tethered at the N- or C-terminus of the isopeptag. Furthermore, we demonstrated site-specific protein immobilization mediated by the reconstitution of split-Spy0128 and isopeptag. In this case, a split-Spy0128 mutant with a free N-terminal Cys residue was first immobilized onto beads chemically modified with a maleimide group through a Michael addition process. Then, an isopeptagged protein was successfully immobilized onto the split-Spy0128-immobilized beads. These results suggest that Spy0128 is a potent proteinaceous scaffold available for bioconjugation both in solution and at a solid surface.

Toward the alignment of conjugated polymers into anisotropically-ordered structure

Recently, oriented polymers and/or polymer nanostructures have attracted a large amount of attention for their potential applications as, for example, electric devices such as FETs, LEDs, photovoltaic cells, and so forth. Besides several methods for organizing conjugated polymers, we have recently reported a novel concept for the alignment of conjugated polymers through the action of supramolecular bundling molecules. We here introduce two different approaches for aligning conjugated polymers; one is to utilize a crosslinking molecule (‘aligner’), and the other is to use a twining polymer (‘twimer’) for organizing conjugated polymers. Aligner binds and crosslinks conjugated polymers in a positive allosteric manner to form organized supramolecular assemblies in solution. The cast film from the solution resulted in a crystalline sheet with periodicities corresponding to the distance between polymers, confirmed by several microscopic studies. On the other hand, twimer helically twins around the conjugated polymers to form one-dimensional (1D) complex in solution and the complex self-assembles into two-dimensionally (2D) aligned structure in drying process. The resulting film of conjugated polymer–twimer composites gave also highly ordered, crystalline structure. The principal results obtained both in solution and solid states and their future perspectives are discussed here.

Solid-in-oil nanodispersions for transdermal drug delivery systems.

Transdermal administration of drugs has advantages over conventional oral administration or administration using injection equipment. The route of administration reduces the opportunity for drug evacuation before systemic circulation, and enables long‐lasting drug administration at a modest body concentration. In addition, the skin is an attractive route for vaccination, because there are many immune cells in the skin. Recently, solid‐in‐oil nanodisperison (S/O) technique has demonstrated to deliver cosmetic and pharmaceutical bioactives efficiently through the skin. S/O nanodispersions are nanosized drug carriers designed to overcome the skin barrier. This review discusses the rationale for preparation of efficient and stable S/O nanodispersions, as well as application examples in cosmetic and pharmaceutical materials including vaccines. Drug administration using a patch is user‐friendly, and may improve patient compliance. The technique is a potent transcutaneous immunization method without needles.