Supramolecular Shear-Thinning Glycopeptide Hydrogels for Injectable Enzyme Prodrug Therapy Applications.

Transplantable catalytic reactors have attracted considerable attention as therapeutic biomedical materials. However, existing transplantable reactors such as biocatalytic films are limited by their invasiveness. Here, we report the fabrication of biocatalytic supramolecular hydrogels via self-assembly of amphiphilic glycopeptides. We show that the hydrogels have shear-thinning properties, demonstrating their potential to be administered using a syringe. Enzymes can be loaded into the hydrogels by simply adding enzyme solution, and the enzyme-loaded hydrogels can transform a prodrug into an anticancer drug that inhibits tumor cell growth. This study demonstrates the potential of these biocatalytic hydrogels as injectable therapeutic reactors for enzyme prodrug therapy.

Self-Assembly of Surfactin into Nanofibers with Hydrophilic Channels in Nonpolar Organic Media.

We investigated the self-assembly of surfactin (SFNa), a cyclic peptide amphiphile produced by Bacillus subtilis, in a nonpolar organic solvent, namely, cyclohexane (CHx). The CHx solution of SFNa formed a thermoreversible organogel. Transmission electron microscopy and small-angle X-ray scattering (SAXS) analyses showed that gelation of the CHx solution of SFNa was caused by physical cross-linking of SFNa nanofibers. Wide-angle X-ray diffraction and Fourier-transform infrared analyses showed that the SFNa nanofibers were formed by one-dimensional stacking of SFNa rings with a period of 0.48 nm corresponding to the length of inter-ring hydrogen bonds between amide groups. A combination of SAXS and small-angle neutron scattering investigations of CHx and deuterated CHx solutions of SFNa nanofibers containing H2O or D2O showed that the SFNa nanofibers had a hydrophilic interior and formed water channels by water incorporation in this region.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity.

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

Delivery of external proteins into the cytoplasm using protein capsules modified with IgG on the surface, created from the amphiphilic two helix-bundle protein OLE-ZIP.

This study explores a new method for delivering therapeutic proteins into specific cells using OLE-ZIP capsules that present IgG. OLE-ZIP capsules is a spherical caspules prepared from amphihilic dimetic coiled-coil peptide, OLE-ZIP. Upon presenting cetuximab, these capsules showed preferential uptake in A431 cells and increased cytotoxicity when loaded with RNase A.

Hydrophobic molecules infiltrating into the poly(ethylene glycol) domain of the core/shell interface of a polymeric micelle: evidence obtained with anomalous small-angle X-ray scattering.

Polymeric micelles have been extensively studied as nanoscale drug carriers. Knowing the inner structure of polymeric micelles that encapsulate hydrophobic drugs is important to design effective carriers. In our study, the hydrophobic compound tetrabromocathecol (TBC) was chosen as a drug-equivalent model molecule. The bromine atoms in TBC act as probes in anomalous small-angle X-ray scattering (ASAXS) allowing for its localization in the polymeric micelles whose shape and size were determined by normal small-angle X-ray scattering (SAXS). Light scattering measurements coupled with field flow fractionation were also carried out to determine the aggregation number of micelles. A core-corona spherical model was used to explain the shape of the micelles, while the distribution of bromine atoms was explained with a hard-sphere model. Interestingly, the radius of the spherical region populated with bromine atoms was larger than the one of the sphere corresponding to the hydrophobic core of the micelle. This result suggests that the TBC molecules infiltrate the PEG hydrophilic domain in the vicinity of the core/shell interface. The results of light scattering and SAXS indicate that the PEG chains at the shell region are densely packed, and thus the PEG domain close to the interface has enough hydrophobicity to tolerate the presence of hydrophobic compounds.

Deconstruction of sulfonamide inhibitors of the urotensin receptor (UT) and design and synthesis of benzylamine and benzylsulfone antagonists.

Potent small molecule antagonists of the urotensin receptor are described. These inhibitors were derived via systematically deconstructing a literature inhibitor to understand the basic pharmacophore and key molecular features required to inhibit the protein receptor. The series of benzylamine and benzylsulfone antagonists herein reported display a combination of nanomolar molecular and cellular potency as well as acceptable in vitro permeability and metabolic stability.

Access to different nanostructures via self-assembly of thiourea-containing PEGylated amphiphiles.

Readily water-soluble PEGylated amphiphiles containing bis-thiourea-based molecular recognition units at the interface of hydrophobic and hydrophilic blocks are developed. Self-assembly of these amphiphiles is found to be dependent on the exact chemical composition of the hydrophobic component. Elongated, spherical, and disk-like micelles are formed with the change in hydrophobic group from stearyl (2A), oleyl (2B), and dodecanol (2C), respectively. The length of the rod-like elongated micelles formed by 2A could be tuned by thermal treatment as well. Synthesis and detailed structural characterization of these amphiphiles by TEM, DSC, synchrotron SAXS techniques are reported. Organic solvent-free direct aqueous encapsulation of doxorubicin, an anticancer drug into these nanostructures is demonstrated.

Drug-Loaded Biocompatible Chitosan Polymeric Films with Both Stretchability and Controlled Release for Drug Delivery.

Chitosan is a natural polysaccharide with the advantageous qualities of biocompatibility and biodegradability, and it has recently been spotlighted as a soft material for a sustainable society. Advantages such as these are in demand for application in various biomaterials. Although extensive studies have been conducted on the preparation of chitosan films, overcoming the problems of weak mechanical properties remains a significant barrier. In the present study, we developed stretchable doxorubicin-loaded biocompatible chitosan films by adding acetic acid in controlled concentrations. The stretchable properties of doxorubicin-loaded chitosan film at various concentrations of acetic acid were measured. Elongation to the point of breakage reached 27% with a high concentration of acetic acid, which could be described as high stretchability. The release ratio of doxorubicin from chitosan film reached 70% with a high acetic acid concentration. The cytotoxicity of doxorubicin-loaded chitosan films was measured, and cancer spheroids had completely collapsed after 7 days. According to the results of skin permeability testing, use of the doxorubicin-loaded chitosan film is a plausible choice for a drug sealant.

A stimulus-responsive shape-persistent micelle bearing a calix[4]arene building block: reversible pH-dependent transition between spherical and cylindrical forms.

A series of cationic calix[4]arene-based lipids with alkyl chains of varying length were newly synthesized, and the ones with propyl and hexyl tails, denoted by CaL[4]C3 and C6, respectively, were found to form spherical micelles at low pH (protonated state of the amine headgroup). Upon deprotonation with increasing pH, CaL[4]C3 showed a sphere-to-cylinder transition, while CaL[4]C6 changed from sphere, to cylinder, to monolayer vesicle. Synchrotron small-angle X-ray scattering (SAXS) patterns from both spherical and cylindrical CaL[4]C3 micelles exhibited a sharp intensity minimum, indicating shape monodispersity. The monodispersity of the CaL[4]C3 spherical micelles was further confirmed by analytical ultracentrifugation (AUC). SAXS, AUC, and static light scattering agreeingly indicated an aggregation number of 6. In contrast, CaL[4]C6 exhibited polydispersity with an average aggregation number of 12. When the number of carbons of the alkyl chain was increased to 9 (CaL[4]C9), cylinder formed at low pH, while at high pH, no clear morphology could be observed. The present results indicate that a very precise combination of tail length, head volume, and rigidity of the building block is required to produce shape-persistent micelles and that the shape-persistence can be maintained upon a structural transition. An attempt to reconstruct a molecular model for the spherical CaL[4]C3 micelle was made with an ab initio shape determining program.

Complex formation of potassium salt of highly fatty acid with hemagglutinin protein in influenza virus via exothermic interaction.

In our previous study, we found highly fatty acid salts, which are a skin-friendly soaps, had a high ability to inactivate the influenza virus. In order to elucidate the mechanism of inactivation of influenza virus, we investigated interactions and complex formation of potassium tetradecanoate (C14K) as a highly fatty acid salt with a virus particle (VP) derived from avian influenza virus by using isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). ITC showed C14K attractively interacted with hemagglutinin protein (HA) which exists in the envelop of VP. SAXS analyses revealed C14K formed highly ordered complex with HA through the attractive interaction. Since the HA is responsible for cell entry events, inactivation of influenza viruses by highly fatty acid salts are derived owing to HA inhibition of influenza viruses through the complex formation. Time-resolved SAXS measurements elucidated the complex formation was completed within 40 s after mixing aqueous solutions of C14K and VP. This result strongly suggests that hand-washing with a highly fatty acid salts is an effective measure to prevent infection with influenza virus without causing rough hands.

Effect of Molecular Architecture on Associating Behavior of Star-Like Amphiphilic Polymers Consisting of Plural Poly(ethylene oxide) and One Alkyl Chain.

Associating behavior of star-like amphiphilic polymers consisting of two or three poly(ethylene oxide) (PEO) chains and one stearyl chain (C18) was investigated. Although the aggregation number (Nagg) of linear analogue of amphiphilic polymers monotonically decreased with increasing number-average molecular weight of PEO (Mn,PEO), the Nagg of micelles of star-like amphiphilic polymers with Mn,PEO = 550 g/mol was smaller than that with Mn,PEO = 750 g/mol, whereas that with Mn,PEO ≥ 750 g/mol showed general Mn,PEO dependence. Small-angle X-ray scattering analyses revealed that the occupied area of one PEO chain on the interface between hydrophobic core and corona layer in the micelles of star-like polymers was much narrower than that in the linear amphiphilic polymers. This result indica ted the PEO chains of star-like polymers partially took unfavorable conformation near the core-corona interface in polymer micelles. The effect of local conformation of PEO chains near the interface on the associating behavior became significant as Mn,PEO decreased. Therefore, in polymer micelles of star-like amphiphilic polymers containing PEO with Mn,PEO = 550 g/mol, the enlargement of occupied area of PEO on the core-corona interface should be caused to avoid the formation of unfavorable conformations of partial PEO chains, resulting in a decrease in Naggs.

Encapsulation of a hydrophobic drug into a polymer-micelle core explored with synchrotron SAXS.

Synchrotron small-angle X-ray scattering (SAXS) at the SPring-8 40B2 and 45XU beamlines was carried out on aqueous solutions of (PEG-P(Asp(Bzl))): partially benzyl-esterified poly(ethylene glycol)-block-poly(aspartic acid) with LE540 loaded up to 8.3 wt %, where LE540 is a very hydrophobic retinoid antagonist drug. The scattering profiles showed characteristic features for core-shell spherical micelles, confirming that P(Asp(Bzl)) forms a hydrophobic core and PEG forms a hydrophilic shell. Before the addition of LE540, a diffraction peak was observed around q = 4 nm(-1), where q is the magnitude of the scattering vector. This peak can be attributed to ordering between alpha-helices made of P(Asp(Bzl)), the so-called nonspecific hexatic arrangement. The P(Asp(Bzl)) helices disappeared as LE540 was added. This result can be interpreted by assuming a uniform distribution of LE540 in the core. By use of a core-shell spherical micelle model, the SAXS data could be well fitted for all of the samples. The analysis indicated that the core radius increases sigmoidally from 5.9 to 6.9 nm upon addition of LE540 whereas the shell radius stayed at 12.5-12.8 nm. The aggregation number that is the average number of PEG-P(Asp(Bzl))'s consisting of one micelle slightly increased from 145 to 182.

Scattering Form Factor of Block Copolymer Micelles with Corona Chains Discretely Distributed on the Core Surface.

Small-angle scattering is a powerful tool to investigate micellar structure, and a model form factor proposed by Pedersen and Gerstenberg [Pedersen, J. S.; Gerstenberg, M. Macromolecules 1996, 29, 1363-1365] has been used quite frequently to analyze experimentally obtained scattering data of block copolymer micelles. Their model consists of a spherical core and the Gaussian corona chains attached to the core surface; the corona chains are considered to be approximately continuously (evenly) distributed on the core surface. In this paper, we present a micellar form factor model in which the corona chains are discretely distributed on the core surface. Our proposed discrete model was found to deviate from the Pedersen-Gerstenberg model as well as another model [Svaneborg, C.; Pedersen, J. S. Phys. Rev. E 2001, 64, R01802.], which incorporates the approximate interference effect between the corona chains, in conditions in which the scattering from the corona chains is stronger than that from a core with lower number density of the corona chains.

Flexibility and cross-sectional structure of an anionic dual-surfactant wormlike micelle explored with small-angle X-ray scattering coupled with contrast variation technique.

The contrast variation technique by adding sucrose to aqueous solvents as an electron density adjusting reagent was applied to small-angle X-ray scattering (SAXS) from a dual surfactant wormlike micelle consisting of sodium lauryl ether sulfate and coconut fatty acid amido propyl betaine in order to evaluate the flexibility and the cross-sectional structure of the micelle. The salt concentration dependence of the zero-shear and dynamic viscosities were found not to be affected by the cation species nor the presence of sucrose in the solution. SAXS showed that the scattering profile [I(q)] can be fitted a double layer cylinder model. By systematically changing the sucrose concentration (CS), a "matching composition" was found in which the shell layer can become invisible in SAXS for each sodium cation concentration ([Na+]). From this composition, the electron densities of the shell (rhoS) and core (rhoC) layers were evaluated to be 370 and 216 e/nm(-3), respectively. These values were consistent with the chemical structure of the surfactant. At these matching compositions, I(q) was measured at low q range (0.03 nm(-1)1.02 M. In these larger [Na+], the I(q) showed an up-turn deviation from the rigid rod limit (i.e., I(q) proportional, variant q(-1)) at low q, indicating appearance of the flexibility of the cylinder. By applying a wormlike cylinder theory, the Kuhn statistical segment length was evaluated as a function of [Na+]. At the infinite limit of [Na+], the intrinsic Kuhn length (excluding of the electrostatic interactions) can be evaluated to be about 20-40 nm. This value was comparable with that of the wormlike micelles made of nonionic surfactants.

Effect of Hydration in Corona Layer on Structural Change of Thermo-Responsive Polymer Micelles.

The effect of hydration in corona layer on temperature responsiveness of polymer micelles consisting of poly(N-vinyl pyrrolidone)-block-poly(n-octadecyl acrylate) (PVP-b-PODA) was investigated. Small-angle X-ray scattering and dynamic light scattering showed two-step shape change of PVP-b-PODA micelles around 45 and 65 °C with elevating temperature, although only one-step shape change was observed at 45 °C in cooling process. In the first step, shape of PVP-b-PODA micelles was changed from disk to ellipsoidal oblate at the melting temperature (Tm) of PODA, although similar micelles consisting of another amphiphilic block copolymers containing PODA simply changed from disk to sphere at the Tm with elevating temperature. PVP-b-PODA micelles changed to spherical shape above 65 °C. Two-dimensional (2D) ¹H-NMR showed the PVP chains were perfectly dehydrated above 65 °C. Therefore, it was suggested that the appearance of ellipsoidal shape between Tm of PODA and 65 °C was caused owing to shape memory effect of pseudo network of corona layer due to robust hydration of PVP chains.

Elucidation of Spatial Distribution of Hydrophobic Aromatic Compounds Encapsulated in Polymer Micelles by Anomalous Small-Angle X-ray Scattering.

Spatial distribution of bromobenzene (BrBz) and 4-bromophenol (BrPh) as hydrophobic aromatic compounds incorporated in polymer micelles with vesicular structure consisting of poly(ethylene glycol)-b-poly(tert-butyl methacrylate) (PEG-b-PtBMA) in aqueous solution is investigated by anomalous small-angle X-ray scattering (ASAXS) analyses near Br K edge. Small-angle X-ray scattering (SAXS) intensities from PEG-b-PtBMA micelles containing BrBz and BrPh were decreased as the energy of incident X-ray approached to Br K edge corresponding to the energy dependence of anomalous scattering factor of Br. The analysis for the energy dependence of SAXS profiles from the PEG-b-PtBMA micelles containing BrBz revealed that BrBz molecules were located in hydrophobic layer of PEG-b-PtBMA micelles. On the contrary, it was found by ASAXS that BrPh existed not only in the hydrophobic layer but also in the shell layer. Since ASAXS analysis successfully accomplished to visualize the spatial distribution of hydrophobic molecules in polymer micelles, it should be expected to be a powerful tool for characterization of drug delivery vehicles.

Inactivation of human and avian influenza viruses by potassium oleate of natural soap component through exothermic interaction.

An influenza epidemic is still a problem despite the development of vaccines and anti-influenza drugs. Preventive measures such as handwashing are fundamental and important for counteracting influenza virus infection. In this study, we clarified the anti-influenza virus effects of surfactants, which are the main components of hand soaps for hand washing: potassium oleate (C18:1), sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS). For a human influenza virus strain (H3N2), C18:1 reduced the infectivity by 4 logs or more, whereas LES and SDS reduced the infectivity by 1 log or less. Similar results were obtained when an avian influenza virus strain (H5N3) was used. The interaction between the surfactant and virus was then investigated by isothermal titration calorimetry. The LES-virus system showed a positive value of enthalpy changes (ΔH), meaning an exothermic interaction that indicated a hydrophobic interaction. In contrast, both the C18:1-virus system and the SDS-virus system showed negative values of ΔH, meaning an endothermic interaction that indicated an electrical interaction. The ΔH value of the C18:1-virus system was much higher than that of the SDS-virus system. A mixture of C18:1 and HA proteins similarly showed negative values of ΔH. These results indicate that influenza virus inactivation by a hydrophobic interaction of a surfactant with the viral envelope is insufficient to prevent infection, whereas inactivation by an electrical interaction of a surfactant with HA proteins is sufficient to prevent influenza virus infection.

Platonic Micelles: Monodisperse Micelles with Discrete Aggregation Numbers Corresponding to Regular Polyhedra.

The concept of micelles was first proposed in 1913 by McBain and has rationalized numerous experimental results of the self-aggregation of surfactants. It is generally agreed that the aggregation number (Nagg) for spherical micelles has no exact value and a certain distribution. However, our studies of calix[4]arene surfactants showed that they were monodisperse with a defined Nagg whose values are chosen from 6, 8, 12, 20, and 32. Interestingly, some of these numbers coincide with the face numbers of Platonic solids, thus we named them "Platonic micelles". The preferred Nagg values were explained in relation to the mathematical Tammes problem: how to obtain the best coverage of a sphere surface with multiple identical circles. The coverage ratio D(N) can be calculated and produces maxima at N = 6, 12, 20, and 32, coinciding with the observed Nagg values. We presume that this "Platonic nature" may hold for any spherical micelles when Nagg is sufficiently small.

Small-angle X-ray scattering from a dual-component organogel to exhibit a charge transfer interaction.

The structure of a dual-component organogel consisting of methyl 4,6-O-(p-aminobenzylidene)-alpha-D-glucopyranoside and methyl 4,6-O-(p-nitrobenzylidene)-alpha-D-glucopyranoside in diphenyl ether was investigated with small-angle X-ray scattering (SAXS). The individual components gelatinized the solvent to yield a colorless gel and the gel fiber consisted of the crystal, providing the crystalline peaks at the same diffraction angles as those of the solid samples. When the components were mixed in equimolar ratio and dissolved in diphenyl ether, a yellow gel was formed and the crystalline peaks disappeared. For all compositions, the SAXS profiles were well fitted by a cylinder model. The cross-sectional radius of gyration, r(c), was determined from the cross-sectional Guinier plot (qI vs q(2), where I and q are the scattering intensity and the magnitude of the scattering vector). The value of r(c) reached a minimum of 3.0 nm at the equimolar composition. By correcting the data for the thermal scattering background, we obtained the entire SAXS profile for the equimolar dual-component gel. From this profile, the radial electron density distribution was determined and the radius of the cylinder was estimated to be 2.6 nm. The electron density distribution thus obtained revealed that four gelator molecules are packed in the sectional direction. This model was consistent with the size of the gelator molecules.

X-ray scattering from immunostimulatory tetrapod-shaped DNA in aqueous solution to explore its biological activity-conformation relationship.

We carried out synchrotron X-ray scattering experiments from four DNA supermolecules designed to form tetrapod shapes; these supermolecules had different sequences but identical numbers of total base pairs, and each contained an immunostimulatory CpG motif. We confirmed that the supermolecules did indeed form the expected tetrapod shape. The sample that had the largest radius of gyration (Rg) induced the most cytokine secretion from cultured immune cells. Structural analysis in combination with a rigid tetrapod model and an atomic scale DNA model revealed that the larger Rg can be ascribed to dissociation of the DNA double strands in the central connecting portion of the DNA tetrapod. This finding suggests that the biological activity is related to the ease with which single DNA strands can be formed.