Yusuke Sanada

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.

Preparation and characterization of polyion complex micelles with phosphobetaine shells.

A pair of oppositely charged diblock copolymers, poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMPC-b-PMAPTAC) and poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PMPC-b-PAMPS), was prepared via reversible addition-fragmentation chain transfer radical polymerization using a PMPC-based macro chain transfer agent. The pendant phosphorylcholine group in the hydrophilic PMPC block has anionic phosphate and cationic quaternary amino groups, which are neutralized within the pendant group. Therefore, the mixing of aqueous solutions of PMPC-b-PMAPTAC and PMPC-b-PAMPS leads to the spontaneous formation of simple core-shell spherical polyion complex (PIC) micelles comprising of a segregated PIC core and PMPC shells. The PIC micelles were characterized using (1)H NMR spin-spin (T2) and spin-lattice relaxation times (T1), diffusion-ordered NMR spectroscopy, static light scattering, dynamic light scattering (DLS), and transmission electron microscopy techniques. The hydrodynamic size of the PIC micelle depended on the mixing ratio of PMPC-b-PMAPTAC and PMPC-b-PAMPS; the maximum size occurred at the mixing ratio yielding stoichiometric charge neutralization. The PIC micelles disintegrated to become unimers with the addition of salts.

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.

β-1,3-d-Glucan Schizophyllan/Poly(dA) Triple-Helical Complex in Dilute Solution

A certain length of poly(deoxyadenylic acid) (dA(X)) can form a novel complex with β-1,3-D-glucan schizophyllan (SPG) with a stoichiometric composition of one dA binding two main chain glucoses. We measured dilute solution properties for the complex with light and small-angle X-ray scattering as well as intrinsic viscosity and found that the complex behaves as a semiflexible rod without branching or cross-linking. We analyzed the data with the wormlike cylinder model, and the chain dimensions and the persistence length for the complexes were consistently determined. The chain flexibility was reduced to almost 25% upon complexation for dA/SPG and to 15% for S-dA/SPG, where S-dA denotes the phosphorothioated DNA analogue. The changes in the molar mass per unit length and the diameter indicated that the helix was elongated or stretched along the axis direction upon the complexation.

Composition dependence of the micellar architecture made from poly(ethylene glycol)-block-poly(partially benzyl-esterified aspartic acid).

Poly(ethylene glycol)-block-poly(partially benzyl-esterified aspartic acid), denoted by PEG-P(Asp(Bzl)), is one of the most examined blockcopolymers for drug carriers. However, little is known about fundamental physical properties. Nine samples of PEG-P(Asp(Bzl)) with different benzylation fractions (F(Bzl)) and aspartic chain lengths (DP(Asp)) were synthesized, and the aggregation number (N(agg)), core radius (R(C)), and other structural parameters were determined with combination of light scattering and synchrotron X-ray small-angle scattering. The major factor to determine N(agg) and R(C) was found to be F(Bzl), i.e., the hydrophobic nature of the core, even though F(Bzl) was changed in the relatively small composition range from 66 to 89 mol %. When we compared the data for the same F(Bzl), the scaling theory was consistent with the core chain length dependence of both core and micelle sizes. The overcrowding nature of the tethered PEG chains on the micelles was increased about 1.3-2.9 times with increasing N(agg) compared with the unperturbed state in solutions.

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.

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.

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.

Naphthalene-hydrophobized β-1,3-glucan nanogel for doxorubicin delivery to immunocytes.

A water soluble β-1,3-glucan schizophyllan (SPG) can be recognized by an immunocyte receptor called dectin-1. When we introduced naphthalene into the side chain of SPG (nSPG), it formed nanogel by physical cross-link and gained capability to ingest hydrophobic compounds such as doxorubicin. Our in vitro assay revealed that this nanogel can be used as specific delivery of anti-cancer drugs to immunocytes.

Association Behavior of Poly(ethylene oxide)–Poly(propylene oxide) Alternating Multiblock Copolymers in Water toward Thermally Induced Phase Separation

Thermal changes in the association behavior of poly(ethylene oxide)-poly(propylene oxide) alternating multiblock (PEO-PPO AMB) copolymers in water are investigated by the use of transmittance and light scattering measurements. Two PEO-PPO AMB copolymers with a different weight fraction of PEO, (EO220PO33)8 and (EO68PO33)9, are prepared. The weight-average molecular weights of (EO220PO33)8 and (EO68PO33)9 estimated by static light scattering measurements are 1.3 × 105 and 4.1 × 104 g mol-1, respectively. The number of PEO-PPO repeating pairs is over 8. It is found that the aqueous solution of (EO220PO33)8 undergoes phase separation with a lower critical solution temperature (LCST) of around 58 °C at 0.3 wt %. For the aqueous (EO68PO33)9 solution, the LCST is estimated to be ca. 42 °C. The critical solution concentration for (EO68PO33)9 is not clear because of a small concentration dependence of Tc at a higher concentration range. Dynamic light scattering measurements indicate that a micellelike aggregate is formed below the LCST. From the Debye plot, it is elucidated that the second virial coefficient, A2, starts going down at around 32 °C for (EO220PO33)8 and below 15 °C for (EO68PO33)9. The A2 value of (EO220PO33)8 approaches 0 near 50 °C, whereas that of (EO68PO33)9 approaches 0 at around 35 °C. At a high temperature, the attractive interaction among the copolymers becomes dominant, thereby inducing the formation of micellelike aggregates.