A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures.

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.

PLA- and PLA/PLGA-Emulsion Composite Biomaterial Sheets for the Controllable Sustained Release of Hydrophilic Compounds.

In the present study, by spin-coating a solution containing w/o (water-in-oil) emulsions and hydrophobic polymers, we obtained sheets possessing uniformly dispersed w/o emulsions. We performed release experiments for more than 100 days and clarified the effects of the number of layers, the sheet-forming polymers (polylactide (PLA), poly(lactic-co-glycolic acid (PLGA)), the ratio of organic solvent to water, and the composition of block copolymers on the release properties of the sheets. For a variety of sheets, we successfully achieved the sustained release of compounds from the sheets for 100⁻150 days. The sustained-release of compounds occurred because the compounds had to diffuse into polymer networks after their release from the emulsions. Interestingly, we observed an inflection point in the release profiles at around 50 days; that is, the sheet exhibited a "two-step" release behavior. The results obtained in the present study provide strong evidence for the future possibility of the time-programmed release of multiple compounds from sheets.

Successful PEGylation of hollow encapsulin nanoparticles from Rhodococcus erythropolis N771 without affecting their disassembly and reassembly properties.

We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent. The nanoparticle obtained in the present study has the potential to incorporate hydrophilic compounds in the internal cavity of the particle by reversibly controllable disassembly and reassembly. The hollow PEGylated encapsulin nanoparticle can be used as a drug carrier for the delivery of hydrophilic biopolymers in future medical applications.

Dual drug release from hydrogels covalently containing polymeric micelles that possess different drug release properties.

In the present study, we designed hydrogels for dual drug release: the hydrogels that covalently contained the polymeric micelles that possess different drug release properties. The hydrogels that were formed from polymeric micelles possessing a tightly packed (i.e., well-entangled) inner core exhibited a higher storage modulus than the hydrogels that were formed from the polymeric micelles possessing a loosely packed structure. Furthermore, we conducted release experiments and fluorescent observations to evaluate the profiles depicting the release of two compounds, rhodamine B and auramine O, from either polymeric micelles or hydrogels. According to our results, (1) hydrogels that covalently contains polymeric micelles that possess different drug release properties successfully exhibit the independent release behaviors of the two compounds and (2) fluorescence microscopy can greatly facilitate efforts to evaluate drug release properties of materials.