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.

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Various cholesterol-bearing pullulans (CHPs) with different molecular weights of the parent pullulan and degrees of substitution (DS) of the cholesteryl moiety were synthesized. The structural characteristics of CHPs in water were studied by static (SLS) and dynamic light scattering (DLS) and the fluorescence probe method. Irrespective of the molecular weight of the parent pullulan and the DS, all of CHPs provided unimodal and monodisperse self-aggregates in water. The size of the self-aggregate decreased with an increase in the DS of the cholesteryl moiety (hydrodynamic radius, 8.4−13.7 nm). However, the aggregation number of CHP in one nanoparticle was almost independent of the DS. The polysaccharide density within the self-aggregate (0.13−0.50 g/mL) was affected by both the molecular weight and the DS of CHPs. The mean aggregation number of the cholesteryl moiety (3.5−5.7), which was estimated by the fluorescence quenching method using pyrene and cetylpyridinium chloride, was almost same for all the CHP self-aggregates. The CHP self-aggregate is regarded as a hydrogel nanoparticle, in which pullulan chains are cross-linked noncovalently by associating cholesteryl moieties. The microenvironment inside or the structural characteristic of the self-aggregate was spectrometrically studied using a fluorescence probe, ANS. The characteristic temperature to cause a structural change of the nanoparticle (T*) decreased with an increase in the DS of CHP and the ionic strength of the medium. The thermoresponsiveness of the nanoparticle hydrogel is related to the partial dehydration of the hydrophobized pullulan upon heating.

Macromolecular complexation between bovine serum albumin (BSA) and self-assembled hydrogel nanoparticle formed by the self-aggregation of cholesterol-bearing pullulan (CHP) was studied by high performance size exclusion column chromatography (HPSEC) and circular dichroism (CD). The CHP self-aggregates complexed with one BSA molecule to give colloidally stable nanoparticles (R G = 17 nm) at pH 7.0 and 25 °C. This was almost irrespective of the substitution degree of the cholesterol group of CHP. The helical content of BSA decreased upon complexation. Unfolding of BSA by either heating or a denaturant such as urea was largely suppressed upon complexation. BSA would be incorporated inside into the hydrogel matrix of the CHP nanoparticle. Kinetic analysis of the complexation suggested a two-step process:  namely, the fast pre-equilibrium of looser binding of BSA to the CHP self-aggregate followed by the slower process of tighter inclusion into the hydrogel network. The substitution degree of the cholesterol group in CHP significantly affected the complexation kinetics.

We have been studying the formation of hydrogel nanoparticles by the self-aggregation of hydrophobized polysaccharide and the effective complexation between these nanoparticles as a host and various globular soluble proteins as a guest. This paper describes a new finding that refolding of the heat-denatured enzyme effectively occurs with the nanoparticles and beta-cyclodextrin according to a mechanism similar to that of a molecular chaperone. In particular, the irreversible aggregation of carbonic anhydrase B (CAB) upon heating was completely prevented by complexation between the heat-denatured enzyme and hydrogel nanoparticles formed by the self-aggregation of cholesteryl group-bearing pullulan (CHP). The complexed CAB was released by dissociation of the self-aggregate upon the addition of beta-cyclodextrin. The released CAB refolded to the native form, and almost 100% recovery of the activity was achieved. The thermal stability of CAB was drastically improved by capture of the unfolded form which was then released to undergo refolding.