Complexation between -chymotrypsin (Chy) and the self-aggregate of cholesterol-bearing pullulan (CHP) was studied by size exclusion column chromatography (SEC), fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC). The CHP self-aggregate strongly complexed with the Chy dimer and formed colloidally stable nanoparticles (Re = 12 nm) at pH 4.2 and 25 °C. Enzymatic degradation of the CHP-Chy complex by pullulanase suggested that Chy may locate deeply inside the matrix of the CHP self-aggregate hydrogel. Upon complexation, the bulk structure of the CHP aggregate changed, the helix content of Chy increased (from 9 to 29%), and the /3-form content decreased (from 34 to 21%). Vmax of the complexed Chy at pH 8.0 decreased up to 1/88 compared with that of free Chy at pH 8.0, while Km did not change much. Chy was released from the complex by the addition of bovine serum albumin (BSA). Released Chy had almost the same enzymatic activity as that of free Chy before the complexation. The secondary structure of Chy in the complex did not change much even after the complex was treated for several hours at 92 °C. Even after the heating, Chy was released from the complex by adding BSA and still had 74% of the original enzyme activity. No thermal unfolding of Chy in the complex was suggested by DSC and fluorescence spectroscopy over the temperature range 20-80 °C. These results indicated that the thermal stability of Chy dramatically increases upon complexation with the CHP self-aggregate.

Thermoresponsive hydrogel nanoparticles were prepared by self-assembly of two different hydrophobically modified polymers, namely a cholesterol-bearing pullulan (CHP) and a copolymer of N-isopropylacrylamide butyl]-N-n-octadecylacrylamide] (PNIPAM-C 18Py). The interactions between CHP and PNIPAM-C18Py were investigated by fluorescence spectroscopy, dynamic light scattering, and size exclusion chromatography. After ultrasonication of a mixture of CHP and PNIPAM-C18Py (5:1 by weight) at 25°C, monodisperse nanoparticles (Dh ) 45 nm) were obtained, consisting of self-assembly of the two polymers associated via their hydrophobic moieties. Evidence from fluorescence and dynamic light scattering demonstrated that, above 32°C, the lower critical solution temperature (LCST) of PNIPAM-C18Py, the colloidal mixed nanoparticles increase in diameter (from 47 to 160 nm), but no macroscopic aggregation could be detected. This phenomenon was thermoreversible: upon cooling the particles recovered their original diameter.

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Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4 + T cells in HLA-A2402-positive patients with therapyrefractory HER2-expressing cancers. Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 Ag of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4 + T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein.Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade1transient skin reaction at the sites of vaccination. HER2-specific CD8 + and/or CD4 + T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom bothT-cell responses were detected in these patients. In four patients with CD8 + T-cell responses, two patients reacted to previously identified HER2 63-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells. Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8 + and/or CD4 + T-cell immune responses.

The Michael reaction of chitosan with acrylic acid was carried out successfully, even in water alone as the reaction medium. As a consequence of its good solubility in water, the reaction product, N‐carboxyethylchitosan, showed excellent biodegradable properties with standard activated sludge.