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In recent years, the fabrication of well-organized proteinosomes has been a popular topic due to the potential applications of the structures in materials science and nanotechnology. A big challenge in the fabrication of proteinosomes is to maintain the structures and the functionalities of proteins on the proteinosomes. In this research, a new concept of polymerization-induced formation of proteinosomes is proposed. In thermal dispersion polymerization of N-isopropyl acrylamide (NIPAM) in the presence of bovine serum albumin (BSA), the growing PNIPAM chains experience phase transition from hydrated coils to dehydrated globules, and the dehydrated PNIPAM chains have hydrophobic interaction with BSA, leading to the formation of hollow proteinosomes. Kinetics studies indicate that there is a transition from the homogeneous polymerization of NIPAM in solution to the heterogeneous polymerization in the proteinosomes. Transmission electron microscopy, atomic force microscopy, confocal laser scanning microscopy and dynamic light scattering all demonstrate the formation of hollow structures. The results of circular dichroism spectroscopy indicate that the secondary structure of BSA remains unchanged in the polymerization process. The formation of proteinosomes is reversible. Upon cooling of the solution to a temperature below the phase transition temperature of PNIPAM, the proteinosomes are dissociated due to the absence of the hydrophobic interaction. The proteinosomes can be used in the encapsulation of hydrophilic compounds in aqueous solution. In this research, not only BSA but also ovalbumin (OVA) is used as a model protein for the fabrication of proteinosomes by the polymerization-induced approach.

Self-assembly of proteins into well-organized proteinosomes has attracted great interest due to the potential medical and biological applications of the structures. Herein, a new concept of electrostatic assisted fabrication of proteinosomes is proposed. The self-assembly is performed by using multi-step dialysis approach, where negatively charged bovine serum albumin-poly(N-isopropylacrylamide) (BSA-PNIPAM) bioconjugate and positively charged enzyme (lysozyme or trypsin) are initially dissolved in phosphate buffer (PB) solution at a high salt concentration, and subsequently the protein solution is dialyzed against PB solutions at low salt concentrations, resulting in the formation of biofunctional proteinosomes. Transmission electron microscopy (TEM), cryo-TEM and light scattering results all demonstrate the formation of hollow structures. The wall of a proteinosome is composed of BSA and enzyme (lysozyme or trypsin), and PNIPAM chains of the bioconjugate are in the corona stabilizing the structure. In comparison with the native enzymes, the enzyme molecules in the assemblies basically retain their bioactivities. The proteinosomes formed by BSA-PNIPAM and lysozyme can be dissociated in the presence of trypsin, and those self-assembled by BSA-PNIPAM and trypsin are able to be self-hydrolyzed, resulting in the dissociation of the structures in aqueous solution. The size and morphology changes of the proteinosomes in the hydrolysis are studied., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Studies on the fabrication of polymer-protein hybrid self-assemblies have aroused great interest over the past years because of a broad range of applications of the materials in drug/protein delivery, biosensors, and enhancement of protein stability. The hybrid assemblies are usually fabricated from polymer-protein bioconjugates, which may suffer from the damages to the protein structures and the loss of functionalities in the synthesis. Herein, we report a simple and efficient approach to the fabrication of vesicle-like structures based on coassembly of homopolymer chains and protein molecules. At room temperature, poly( N -isopropylacrylamide) (PNIPAM) and bovine serum albumin (BSA) are able to form complexes through hydrophobic interactions in aqueous solution. Upon heating to a temperature above the cloud point of PNIPAM, vesicle-like structures with collapsed PNIPAM in the walls and BSA at the surfaces are formed. The size and membrane thickness of the assemblies can be tuned by the molar ratio of PNIPAM to BSA. The hydrophobic interaction between PNIPAM and BSA plays a key role in the complex formation and self-assembly process. The complexes and assembled structures are analyzed by using micro differential scanning calorimetry, light scattering, and transmission electron microscopy. BSA in the assemblies retains over 90% of its activity, and the protein stability is enhanced because of the hydrophobic interaction between proteins and polymers. This approach allows us to prepare polymer-protein assemblies without bioconjugate synthesis. Meanwhile, possible damages to the protein structures and the loss of bioactivities of proteins can be avoided.

Studies on protein adsorption on nanoparticles have attracted great interest over the past years due to the unique properties of the protein-immobilized nanoparticles. However, the effects of protein adsorption on the stability of nanoparticles and the role of hydrophobic interaction in the adsorption have not been fully understood. Herein, fundamental research on protein-induced dissociation of biomolecular assemblies based on hydrophobic interaction is reported. Bovine serum albumin (BSA) is used as a model protein, and cholesterol-glutathione bioconjugate (Ch-GSH) and cholesterol-terminated polyethylene glycol (Ch-PEG) are chosen as model amphiphilic biomolecules. Ch-GSH or Ch-PEG molecules are able to self-assemble into vesicles. The walls and the coronae of the assemblies are composed of hydrophobic Ch and hydrophilic GSH (or PEG), respectively. Upon addition of BSA into phosphate buffer saline solutions of the assemblies, vesicle structures are dissociated and small-sized aggregates composed of BSA, and amphiphilic biomolecules are formed. The dissociation temperatures of the vesicles can be determined by dynamic light scattering. Transmission electron microscopy and size exclusion chromatography are used to demonstrate the dissociation of the assemblies and the formation of aggregates. The hydrophobic interaction between hydrophobic patches on BSA molecules and Ch groups in the walls of the assemblies is responsible for the dissociation of the vesicles and the formation of the aggregates with smaller sizes.

Synthesis and self-assembly of bioconjugates composed of proteins and synthetic molecules have been widely studied because of the potential applications in medicine, biotechnology, and nanotechnology. One of the challenging research studies in this area is to develop organic solvent-free approaches to the synthesis and self-assembly of amphiphilic bioconjugates. In this research, dialysis-assisted approach, a method based on unimer-aggregate equilibrium, was applied in the coassembly of lysozyme and conjugate of cholesterol and glutathione (Ch-GSH). In phosphate buffer solution, amphiphilic Ch-GSH conjugate self-assembles into vesicles, and the vesicle solution is dialyzed against lysozyme solution. Negatively charged Ch-GSH unimers produced in the unimer-vesicle exchange equilibrium, diffuse across the dialysis membrane and have electrostatic interaction with positively charged lysozyme, resulting in the formation of Ch-GSH-lysozyme bioconjugate. Above a critical concentration, the three-component bioconjugate molecules self-assemble into bioactive vesicles.