Complex Consisting of β‑Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the β-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA₄₀ (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA_257–264) and dA₄₀ and made complexes with SPG. The conjugates with a disulfide bond between OVA_257–264 and dA₄₀ were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA–OVA_257–264/SPG and CpG–dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide–dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases

Glutamic Acids Bearing Calix[4]arene Micelles: pH-Controllable Aggregation Number Corresponding to Regular Polyhedra

We have prepared a new calix[4]­arene-based lipid containing glutamic acid as the hydrophilic group. The α-amine and the γ-carboxylic acid groups of the glutamic acid moiety allowed a continuous change in the state of the headgroup from cationic to zwitterionic and then to anionic with increasing pH. Accompanying this headgroup change, micelles of the lipid underwent a morphological transformation from spherical to cylindrical and again to spherical. The morphological transition was ascribed to the change in the lipid conformation corresponding to the pH conditions. Interestingly, at acidic and basic pH, the spherical micelles demonstrated monodispersity in terms of the aggregation number, which agreed with the vertex numbers of Platonic solids, indicating the formation of Platonic micelles. At acidic and basic pH, the lipid conformations were almost identical, but there was a slight difference in the hydrophilic volume, which might affect the packing behavior of the lipid in micelles and account for the difference in the aggregation number. This study clearly demonstrates the precise pH-controllable aggregation number of micelles, which belong to the Platonic micelle systems

Supramolecular Chirality: Vesicle-to-Chiral Helix Transition of the Micelles Consisting of a Sugar-Bearing Calix[4]arene Surfactant

Supramolecular self-assembly and the resulting chiral transfer from the molecular level to the nanoscale is a major topic in modern supramolecular chemistry. We synthesized a galactose-bearing calix[4]­arene surfactant (chiral) and mixed it with a primary amine-bearing analogue (achiral). The mixture showed strong induced circular dichroism (ICD) at an almost 3:2 molar ratio of the two surfactants, and exothermic heat was observed upon mixing. The magnitude of Δ<i>H</i> was comparable to that of van der Waals interactions. This phenomenon indicated that the ICD can be ascribed to the formation of a new supramolecular assembly in which the stoichiometric interaction between the two molecules leads to complexation and the resultant complex has chiral morphology. Transmission electron microscopy and small-angle X-ray scattering showed that the galactose-bearing surfactant forms vesicles, and the mixing induces a transition from the vesicles to threadlike cylinders with a diameter of ∼3.0 nm. We presume that these cylinder are twisted because of chiral transfer from the chiral galactose moiety and show ICD

Rediscovering the Monodispersity of Sulfonatocalix[4]arene-Based Micelles

When the micellar aggregation number (<i>N</i>_agg) is small enough (<30), the <i>N</i>_agg matches the value of vertexes of a regular polyhedron: Platonic solids, and demonstrates perfect monodispersity. These micelles are named Platonic micelles and are particularly found in the system of calix[4]­arene-based micelles due to the rigid structure of the backbone molecule. Although sulfonatocalix[4]­arene-based micelles are among the most studied host molecules in supramolecular chemistry, their micellar properties as Platonic micelles have thus far been overlooked. In this study, we prepared various sulfonatocalix[4]­arene-based amphiphiles bearing alkyl chains with different lengths and investigated their aggregation behavior. When the amphiphiles formed spherical micelles, they demonstrated monodispersity in terms of <i>N</i>_agg, whose value changed from 4 to 17, and then to 24, upon increasing the carbon number in each alkyl chain from C5 to C6, and then to C7, respectively. Although the numbers 17 and 24 do not match the vertices of regular polyhedra, these values can be reasonably explained by the Thomson problem, which considers the Coulomb potential for calculating the best packing on a sphere with multiple identical spherical caps. This study describes rediscovery of the monodispersity of sulfonatocalix[4]­arene-based micelles, which is consistent with the idea of Platonic micelles

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

Adjuvant Activity Enhanced by Cross-Linked CpG-Oligonucleotides in β‑Glucan Nanogel and Its Antitumor Effect

Cancer vaccine has the ability to directly eradicate tumor cells by creating and activating cytotoxic T lymphocytes (CTLs). To achieve efficient CTL activity and to induce Th1 responses, it is essential to administer an appropriate adjuvant as well as an antigen. CpG-ODN is known as a ligand of Toll-like receptor 9 (TLR9) and strongly induces Th1 responses. In our previous study, we developed a CpG-ODN delivery system by use of the formation of complexes between ODN and a β-glucan SPG, denoted as CpG/SPG, and demonstrated that CpG/SPG induces high Th1 responses. In this study, we created a nanogel made from CpG/SPG complexes through DNA–DNA hybridization (cross-linked (CL)-CpG). Immunization with CL-CpG induced much stronger antigen-specific Th1 responses in combination with the antigenic protein ovalbumin (OVA) than that with CpG/SPG. Mice preimmunized with CL-CpG and OVA exhibited a long delay in tumor growth and an improved survival rate after tumor inoculation. These immune inductions can be attributed to the improvement of cellular uptake by the combination of increased size and the cluster effect of the β-glucan recognition site in the nanogel structure. In other words, the particle nature of CL-CpG, instead of the semiflexible rod conformation of CpG/SPG, enhanced the efficacy of a cancer vaccine. The present results indicate that CL-CpG can be used as a potent vaccine adjuvant for the treatment of cancers and infectious diseases

Self-Assembly of Calix[4]arene-Based Amphiphiles Bearing Polyethylene Glycols: Another Example of “Platonic Micelles”

The aggregation number of classical micelles exhibits a certain distribution, which is a recognizable feature of conventional micelles. However, we recently identified perfectly monodisperse calix[4]­arene-based micelles whose aggregation numbers agree with the vertex numbers of regular polyhedra, that is, Platonic solids, and thus they are named “Platonic micelles”. Regarding our hypothesis of the formation mechanism of Platonic micelles, both repulsive interactions including steric hindrance and electrostatic repulsions among the headgroups are important for determining their aggregation number; however, neither of these is necessarily needed to consider. In this study, we employed polyethylene glycols (PEGs) as the nonionic headgroup of calix[4]­arene-based amphiphiles to study the effects of only repulsive interactions caused by steric hindrance on the formation of Platonic micelles. The amphiphiles containing relatively low-molecular-weight PEGs (550 or 1000 g mol^–1) form dodecamer or octamer micelles, respectively, with no variation in the aggregation number. However, relatively high-molecular-weight PEGs (2000 g mol^–1) produce polydispersed micelles with a range of aggregation number. PEG 2000 exhibits a greater affinity for water than PEG 550 and 1000, resulting in fewer hydrophobic interactions in micelle formation, as indicated by the drastic increase of the critical micelle concentration (CMC) value in the PEG 2000 system. The instability of the structure of PEG_2kCaL5 micelles might contribute to the higher mobility of PEG in the micellar shell, resulting in a non-Platonic aggregation number with polydispersity

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

A certain length of poly(deoxyadenylic acid) (dA_<i>X</i>) 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

Core–Shell–Corona Micelles from a Polyether-Based Triblock Terpolymer: Investigation of the pH-Dependent Micellar Structure

Core–shell-corona micelles featuring a pH-responsive shell have been characterized in dilute aqueous solution at different pH values (4–8) by using dynamic light scattering (DLS), field-flow fractionation coupled with multiangle light scattering detector (FFF-MALS), steady-state fluorescence, small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). The micelles are formed by self-assembly of a polyether-based triblock terpolymer consisting of a hydrophobic poly­(<i>tert</i>-butyl glycidyl ether) block (P<i>t</i>BGE), a pH-responsive modified poly­(allyl glycidyl ether) segment (PAGE_COOH), and a neutral hydrophilic poly­(ethylene oxide) block (PEO). Because of the side-chain carboxylic acids in the middle block, the micellar structure and size depends on the solution pH. Hereby, we show that an increase in pH induces a decrease in the aggregation number (<i>N</i>_agg). In addition, the combination of the above measurements revealed an unexpected morphological change from spherical to ellipsoidal micelles by increasing pH

Ultraviolet Patterned Calixarene-Derived Supramolecular Gels and Films with Spatially Resolved Mechanical and Fluorescent Properties

Supramolecular assemblies have in the past been considered mechanically weak and in most cases unable to withstand their own weight. Calixarene-derived networks can, however, provide robust supramolecular gels. Incorporating a photoreactive stilbene moiety, we show that the aggregation state of the material can be tuned by heating and UV exposure in order to control the mechanical as well as the fluorescent properties. Regulating the extent of heating to control the proportion of H-aggregates and J-aggregates and further cross-linking of H-aggregates by control over UV exposure allows for adjustable photopatterning of the fluorescence as well as the material stiffness in the range from ∼100 to 450 kPa. We expect this straightforward supramolecular system will be suitable for advanced prototyping in applications where modulus and shape are important design criteria