Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.
Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-Z E ) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (Z R -ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.
Bacterial adhesion to stainless steel 316L (SS316L), which is an alloy typically used in many medical devices and food processing equipment, can cause serious infections along with substantial healthcare costs. This work demonstrates that nanotextured SS316L surfaces produced by electrochemical etching effectively inhibit bacterial adhesion of both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, but exhibit cytocompatibility and no toxicity toward mammalian cells in vitro. Additionally, the electrochemical surface modification on SS316L results in formation of superior passive layer at the surface, improving corrosion resistance. The nanotextured SS316L offers significant potential for medical applications based on the surface structure-induced reduction of bacterial adhesion without use of antibiotic or chemical modifications while providing cytocompatibility and corrosion resistance in physiological conditions.
We present the effect of molecular weight (MW) of polyelectrolytes (PEs) on the disintegration behavior of weak PE multilayer films consisting of linear poly(ethylene imine) (LPEI) and poly(methacrylic acid) (PMAA). The multilayer films prepared by the spin-assisted layer-by-layer deposition have well-ordered internal structures and also show the linear thickness growth behavior regardless of MWs of PMAA. The well-defined weak PE multilayer films were subject to disintegration into bulk solution when the electrostatic interactions between LPEI and PMAA layers were reduced by treatment at pH 2. However, we demonstrated the change in the disintegration mode and kinetics (i.e., from burst erosion to controlled surface erosion) as a function of MW of PMAA based on neutron reflectivity and quartz crystal microbalance with dissipation, revealing the correlation between the structural changes and the viscoelastic responses of the weak PE films upon pH treatment. Also, the unique swelling behavior as well as the significant increase in dissipation energy was monitored before the complete disintegration of the multilayer films containing high MW PMAA, which is believed to originate from their slow rearrangement kinetics within the film. We believe that the results shown in this study provide chain-level understanding as to the MW-dependence on pH-triggered disintegration mechanism of weak PE multilayer films.
Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on the surface and can also carry protein, small-molecule, or nanoparticle cargo in the vesicle lumen. To create a material with a more complex hierarchical structure, we combined calcium phosphate with zipper fusion proteins containing random coil polypeptides to produce hybrid protein-inorganic supraparticles with high surface area and porous structure. The use of a functional enzyme created supraparticles with the ability to degrade inflammatory cytokines. Our characterization of these protein materials revealed that the molecular interactions are complex because of the large size of the protein building blocks, their folded structures, and the number of potential interactions including hydrophobic interactions, electrostatic interactions, van der Waals forces, and specific affinity-based interactions. It is difficult or even impossible to predict the structures a priori. However, once the basic assembly principles are understood, there is opportunity to tune the material properties, such as size, through control of the self-assembly conditions. Our futur...
Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.
Anti-biofouling has been improved by passive or active ways. Passive antifouling strategies aim to prevent the initial adsorption of foulants, while active strategies aim to eliminate proliferative fouling by destruction of the chemical structure and inactivation of the cells. However, neither passive antifouling strategies nor active antifouling strategies can solely resist biofouling due to their inherent limitations. Herein, we successfully developed multimodal antibacterial surfaces for waterborne and airborne bacteria with the benefit of a combination of antiadhesion (passive) and bactericidal (active) properties of the surfaces. We elaborated multifunctionalizable porous amine-reactive (PAR) polymer films from poly(pentafluorophenyl acrylate) (PPFPA). Pentafluorophenyl ester groups in the PAR films facilitate creation of multiple functionalities through a simple postmodification under mild condition, based on their high reactivity toward various primary amines. We introduced amine-containing poly(dimethylsiloxane) (amine-PDMS) and dopamine into the PAR films, resulting in infusion of antifouling silicone oil lubricants and formation of bactericidal silver nanoparticles (AgNPs), respectively. As a result, the PAR film-based lubricant-infused AgNPs-incorporated surfaces demonstrate outstanding antibacterial effects toward both waterborne and airborne Escherichia coli, suggesting a new door for development of an effective multimodal anti-biofouling surface.
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