Satoru Kidoaki

Time-programmed dual release formulation by multilayered drug-loaded nanofiber meshes

To develop a drug carrier that enables time-programmed dual release in a single formulation, multilayered drug-loaded biodegradable nanofiber meshes were designed using sequential electrospinning with the following construction: (i) first drug-loaded mesh (top), (ii) barrier mesh, (iii) second drug-loaded mesh, and (iv) basement mesh (bottom). The drug release speed and duration were controlled by designing morphological features of the electrospun meshes such as the fiber diameter and mesh thickness. Control of the timed release of the second drug-the retardation period-was accomplished by appropriate design of the barrier mesh thickness. An in vitro release experiment demonstrated that the tetra-layered construction described above with appropriate morphological features of each component mesh can provide timed dual release of the respective drugs. The time-programmed dual release system using the multilayered electrospun nanofiber meshes was demonstrated as a useful formulation for advanced multidrug combination therapy requiring regiospecific administration of different drugs at different times. The potential use of the present multilayered formulation is discussed for application to biochemical modulation as one administrative strategy for use in sequential chemotherapy employing multiple anti-tumor drugs.

Structure of collapsed persistent macromolecule: Toroid vs. spherical globule

We studied theoretically the behavior of a collapsed persistent macromolecule in poor solvent as a model of collapse transition of single double-stranded DNA chain, and constructed the diagram of states in the variables with contour length of a macromolecule and quality of the solvent. We found that the state of toroidal globule exists as an intermediate state between the states of elongated coil state and the spherical globule. Our theoretical result suggests that a single linear macromolecule with a high degree of polymerization can form a toroidal globule. However, the range in which the toroidal structure is stable decreases as the macromolecule length increases. Experimental observation with transmission electron microscopy has been performed to study the globular structure of single DNA chain (bacteriophage T4 DNA, λ-DNA) collapsed by hexammine cobalt (III) at different concentrations. We found that an extremely long chain of T4 DNA (166 kbp), with a contour length of 56 μm, actually forms a toroidal globule, and that isotropic spherical globule appears at higher hexammine cobalt concentration.

Self-organized nanostructures constructed with a single polymer chain

Abstract A Monte Carlo simulation was performed on the folding transition of a single stiff polymer chain. We have adapted a polymer model: neighboring beads are connected by a stick and the length of each stick is taken as 1 8 of the Kuhn length. Among the products of the folding from the single chain, the toroidal structure is found to be the most stable. Rod and fused structures between rod and toroid are also generated as metastable states with almost infinite lifetime. These morphologies resemble the experimental structures observed with electron microscopy for the products of collapse from single duplex DNA chains.

Elasticity boundary conditions required for cell mechanotaxis on microelastically-patterned gels

Directional cell migration induced by a mechanical gradient on a substrate surface toward a harder region, so-called mechanotaxis or durotaxis, has recently drawn attention not only in the field of mechanobiology but also for possible cell manipulation in biomedical engineering. Before we can use mechanotaxis to control cell migration on a biomaterial surface, quantitative design criteria for a microelasticity gradient should be established. To clarify the conditions required to induce mechanotaxis, the effects of a microelasticity boundary on cell culture hydrogels have been systematically assessed with regard to fibroblast migration based on a custom-built reduction projection-type photolithographic microelasticity patterning system with elasticity-tunable photocurable styrenated gelatins, which is a thoroughly-improved system of our previous simple photomasking method [41]. As a result, the conditions required to induce mechanotaxis were found to include a certain threshold jump in elasticity (30-40 kPa) and a sufficiently narrow width of the elasticity boundary (50 μm) comparable to a single cells adhered area, i.e., a sufficiently high gradient strength (30-40 kPa/50 μm in our gelatinous gel system). A significant asymmetric distribution of the number and size of focal adhesions across the elasticity boundary was confirmed to be one of the driving factors of mechanotaxis by indirect immunofluorescence microscopy, and mechanistic considerations in the design criteria are discussed.

Time-dependent complex formation of dendritic poly(L-lysine) with plasmid DNA and correlation with in vitro transfection efficiencies

Dendritic poly(L-lysine) of the 6th generation shows high transfection efficiency into several cultivated cells with low cytotoxicity. In order to understand the mechanism of complex formation with plasmid DNA, the complex was observed using atomic force microscopy. After mixing for 15 min, 1-2 microns assemblies of complexes composed of several small particles (50-200 nm) were observed. At the same time, individual small complexes of 50 to 500 nm were observed on a mica surface. After incubation for 2 h, only the large complexes were found on the mica surface. As a result of further dynamic light scattering analysis and measurement of the transfection efficiency at different time points, the transfection efficiency of KG6 was found to increase with increasing size of the DNA-complexes. This result indicates that large complexes of more than 1 micron are major species that contribute to transfection in vitro.

Reversible hydrogel formation driven by protein-peptide-specific interaction and chondrocyte entrapment.

We developed a hydrogel self-assembling method driven by the interaction between recombinant tax-interactive protein-1 (TIP1) with the PDZ domain in a molecule, which is fused to each end of the triangular trimeric CutA protein (CutA-TIP1), and a PDZ domain-recognizable peptide which is covalently bound to each terminus of four-armed poly(ethylene glycol) (PDZ-peptide-PEG). Genetic manipulation based on molecular-dynamic simulation generated a cell-adhesive RGD tripeptidyl sequence in the CutA loop region [CutA(RGD)-TIP1]. Spontaneous viscoelastic hydrogel formation occurred when either CutA-TIP1- or CutA(RGD)-TIP1-containing buffer solution and PDZ-peptide-PEG-containing buffer solutions were stoichiometrically mixed. Dynamic viscoelasticity measurement revealed shear stress-dependent reversible-phase transformation: a spontaneous viscoelastic hydrogel was formed at low shear stress, but it was transformed into a sol at high shear stress. Upon the cessation of shear, hydrogel was restored. When chondrocytes were pre-mixed with one of these two components containing buffer solutions, the stoichiometric mixed solution was also spontaneously gelled. Individual rounded cells and multicellular aggregates were entrapped within both hydrogels without substantial cellular impairment regardless of the presence or absence of RGD motif in the CutA-TIP1 molecule. The potential use of such a shear-sensitive hydrogel for injectable cell delivery into diseased or lost cartilage tissue is discussed.

Mechanistic aspects of protein/material interactions probed by atomic force microscopy

Abstract Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves ( f – d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f – d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f – d curves.

Phosphorylcholine-endcapped oligomer and block co-oligomer and surface biological reactivity

Phosphorylcholine (PC)-endcapped oligomer and block co-oligomer were prepared by employing a photoiniferter-based quasi-living polymerization technique. The designed oligomer had a PC polar head group attached to an alkylene chain at one end of the molecule and an oligo(styrene) (oligoST) segment at the other end. In the co-oligomer, an oligo(N,N-dimethylacrylamide) (oligoDMAAm) segment was inserted between both ends of the oligomer mentioned above. Surface coating of these amphiphilic substances, using an appropriate coating procedure, resulted in a very hydrophilic characteristic, suggesting that the oligoST anchored on the substrate and the PC polar head group was exposed to or located on the outer coating layer. Non-cell adhesivity in serum-containing medium was observed, while slightly reduced protein adsorption was observed. Thus, PC-endcapped oligomer and block co-oligomer appear to function as a biocompatible coating.

Competition between compaction of single chains and bundling of multiple chains in giant DNA molecules.

It has been established that in a dilute solution individual giant DNA molecules undergo a large discrete transition between an elongated coil state and a folded compact state. On the other hand, in concentrated solutions, DNA molecules assemble into various characteristic states, including multichain aggregate, liquid crystalline, ionic crystal, etc. In this study, we compared single-chain and multiple-chain events by observing individual chains using fluorescence microscopy. We used spermidine, SPD(3+), as a condensing agent for giant DNA. When the concentration of DNA is below 1 microM in base-pair units, individual DNA molecules exhibit a transition from an elongated state to a compact state. When the concentration of DNA is increased to 10 microM, a thick fiberlike assembly of multiple chains appears. AFM measurements of this thick fiber revealed that more than tens of DNA molecules form a bundle structure with parallel ordering of the chains. The transition between single-chain compaction and bundle formation with multiple-chain assemblies was reproduced by a theoretical calculation.

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers.

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor alpha, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.