Supramolecular Assembly between Nanoparticles of Hydrophobized Polysaccharide and Soluble Protein Complexation between the Self-Aggregate of Cholesterol-Bearing Pullulan and .alpha.-Chymotrypsin

Abstract

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.

Controlled release and interaction of protein using self‐assembling peptide RATEA16 nanofiber hydrogels

“…Especially, the hydrophobic interaction between the hydrophobic domain and the hydrophobic surface of insulin exposed by part denaturation is very important, and is the main driving force for the denaturation of the insulin-FITC native structure. 19,24,[29][30][31][32] Possibly due to part denaturation, it was observed that the hydrophilic insulin-FITC molecules immediately became precipitate, when adding peptide RATEA16 into its aqueous solution (see Supporting Information). At the same time, the extent to b-sheet secondary structure of peptide increased, indicating that the existence of insulin-FITC molecules redounded to the b-sheet formation of peptide RATEA16.…”

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

“…CHP can form stable hydrogel NPs by self-assembly, 13,14 and can then form complexes with various drugs and proteins by hydrophobic interaction and release them upon exposure to specific proteins or cyclodextrin. [15][16][17][18] The hydrogel NPs can also be used as a drug carrier system in medicine 17,19,20 as well as artificial molecular chaperones in biotechnology. [21][22][23] On the basis of these, our group developed a new method to synthesize cholesterol-modified pullulan (CHSP) by directly grafting proper cholesterol residues onto pullulan using succinyl linkages, thus providing a new strategy for the effective and safe synthesis of CHSP.…”

In vitro and in vivo evaluation of folate receptor-targeting amphiphilic copolymer-modified liposomes loaded with docetaxel

“…15 Cholesterol is often used to modify biomaterials hydrophobically due to its rigid and highly hydrophobic sterol skeleton. 16,17 The two main goals of the present work were, firstly, to report a novel amphiphilic copolymer, ie, FA-PEG-PCHL, with a series of molecular weights (4374-12394 Da) and low cytotoxicity, and, secondly, to investigate the characteristics, in vitro cytotoxicity, intracellular uptake, apoptotic effect, in vivo pharmacokinetics, and tissue distribution of FA-PEG-PCHL-modified, freeze-dried docetaxel-loaded liposomes (FA-PDCT-L).…”