Solution properties in water of hydrophobized pullulan containing 1.6 cholesterol groups per 100 glucose units (CHP-55-1.6) were studied by size exclusion column chromatography (SEC), dynamic (DLS) and static light scattering (SLS) methods, electron microscopy, lH NMR, and fluorescence spectroscopy.SEC measurementsshow that CHP (l.Omg/mL, 0.lOwt %) intermolecularly aggregates and providesrelatively monodispersive particles upon ultrasonication. Spherical particles with relatively uniform size (the diameter, 25 * 5 nm) were observed in the negatively stained electron microscopy of the aqueous CHP solution. The hydrodynamic radius of the CHP self-aggregate determined by DLS was approximately 13 nm, and the aggregation number determined by SLS was approximately 13; the weight averaged molecular weight of the self-aggregate was 7.6 X lo5, the root mean-square radius of gyration (Re) was 16.8 nm, and the second virial coefficient (Az) was 2.60 X 10-4 (mol mL)/g2. The critical concentration of the self-aggregate formation determined fluorometrically was 0.01 mg/mL. In addition, they showed no surface activity at all up to the concentration of 0.145 mg/mL. Existence of microdomains which consist of both the rigid core of hydrophobic cholesterol and the relatively hydrophilic polysaccharide shell was auggested on the basis of both the line broadening of the proton signal of the cholesterol moiety of CHP(8 = 0.6-2.4 ppm) in the 'H NMR spectrum and the incorporation of several hydrophobic fluorescent probes in the CHP self-aggregates. The CHP self-aggregates strongly complexed with hydrophobic and less hydrophilic fluorescent probes similarly to the case of cyclodextrin.

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

To better understand protein/material and cell/material interactions at the submolecular level, well-defined polymer brushes consisting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) on silicon wafers were prepared by atom transfer radical polymerization (ATRP). Silicon wafers were treated with 3-(2-bromoisobutyryl)propyl dimethylchlorosilane (BDCS) to form a monolayer that acts as initiators for ATRP. Silicon-supported BDCS monolayers were soaked in a methanol/water mixture solution containing Cu(I)Br, bipyridine, and a sacrificial initiator. After MPC was added to the solution, ATRP was carried out for 18 h. The molecular weight and thickness of the PMPC brush layer on the silicon surface increased with an increase in the polymerization time. The dense polymer brushes were obtained by the "grafting from" system. By selective decomposition of the BDCS monolayer by UV light-irradiation, the PMPC brush region and the sizes were well controlled, resulting in fabricating micropatterns of the PMPC brushes. When the thickness of the PMPC brush layer was greater than 5.5 +/- 1.0 nm (3 h polymerization), serum protein adsorption and fibroblast adhesion were effectively reduced, i.e., proteins and cells could recognize such thin polymer brushes on the surface. In addition, the density of the adherent cells on the patterned PMPC brush surface could be controlled by changing the size of the pattern.