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We present the effect of molecularweight (MW) of polyelectrolytes (PEs) on the disintegration behaviorof weak PE multilayer films consisting of linear poly(ethylene imine)(LPEI) and poly(methacrylic acid) (PMAA). The multilayer films preparedby the spin-assisted layer-by-layer deposition have well-ordered internalstructures and also show the linear thickness growth behavior regardlessof MWs of PMAA. The well-defined weak PE multilayer films were subjectto disintegration into bulk solution when the electrostatic interactionsbetween 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 functionof MW of PMAA based on neutron reflectivity and quartz crystal microbalancewith dissipation, revealing the correlation between the structuralchanges and the viscoelastic responses of the weak PE films upon pHtreatment. Also, the unique swelling behavior as well as the significantincrease in dissipation energy was monitored before the complete disintegrationof the multilayer films containing high MW PMAA, which is believedto originate from their slow rearrangement kinetics within the film.We believe that the results shown in this study provide chain-levelunderstanding as to the MW-dependence on pH-triggered disintegrationmechanism of weak PE multilayer films. [ABSTRACT FROM AUTHOR]

We have designed the controlled release platforms basedon polyelectrolyte(PE) blend multilayer films to investigate the release mode and kineticsat the nanoscale level. The model blend multilayer films are composedof positively charged layers with weak polyelectrolytes (PEs) (linearpoly(ethylenimine), LPEI) and negatively charged blend layers withmixtures of strong (poly(sodium 4-styrenesulfonic acid), PSS) andweak (poly(methacrylic acid), PMAA) PEs. The blend multilayer films([LPEI/PSS:PMAA]n) with well-defined internalstructure were prepared by the spin-assisted layer-by-layer (LbL)deposition method. Release properties of the multilayer films weresystematically studied as a function of blend ratio by neutron reflectivity(NR), ellipsometer, AFM, FT-IR spectroscopy, and quartz crystal microbalancewith dissipation (QCM-D). Since PSS strong PEs serve as robust skeletonswithin the multilayer films independent of external pH variation,the burst disruption of pure weak PE multilayer films was dramaticallysuppressed, and the release kinetics could be accurately controlledby simply changing the PSS content within the blend films. These releaseproperties of blend multilayer films form the basis for designingthe controlled release of target active materials from surfaces. [ABSTRACT FROM AUTHOR]

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.

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept(o)ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure-property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections.

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., Competing Interests: The authors declare no competing financial interest.

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 future efforts on the fundamental side will focus on identifying the phase space of self-assembly of these fusion proteins and additional experimental levers with which to control and tune the resulting materials. On the application side, we are investigating an array of different functional proteins to expand the use of these structures in both therapeutic protein delivery and biocatalysis.

Gas bubbles enhance contrast in ultrasound sonography and can also carry and deliver therapeutic agents. The mechanical properties of the bubble shell play a critical role in determining the physical response of gas bubbles under ultrasound insonation. Currently, few methods allow for tailoring of the mechanical properties of the stabilizing layers of gas bubbles. Here, we demonstrate that blending of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic triblock copolymer with a recombinant protein, oleosin, enables the tuning of the mechanical properties of the bubble stabilizing layer. The areal expansion modulus of gas bubbles, as determined by micropipette aspiration, depends on the structure as well as the concentration of PEO-PPO-PEO triblock copolymers. We believe our method of using a mixture of PEO-PPO-PEO and oleosin can potentially lead to the formation of microbubbles with stabilizing shells that can be functionalized and tailored for specific applications in ultrasound imaging and therapy.

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.

We have developed the in situ fibrillation of κ-casein, employed as amyloid precursor, within multilayer films consisting of κ-casein and poly(acrylic acid) (PAA) prepared by the layer-by-layer (LbL) deposition. The fibrillation of κ-casein within the multilayered films is strongly dependent on the extent of intermolecular interactions between κ-casein and PAA. When films constructed initially at pH 3 were heat treated at the same pH, κ-casein did not transform into fibrils. However, when the films were subjected to heat treatment at pH 5, κ-casein was transformed into fibrils within multilayer films due to weakened intermolecular interactions between κ-casein and PAA. We also noted that the multilayer film was swollen at pH 5 by the charge imbalance within the film, which we believe gives enough mobility for κ-caseins to form fibrils with adjacent κ-caseins within the multilayer. The fibrils were found to be uniformly distributed across the entire film thickness, and the aspect ratio as well as the number density of fibrils increased as a function of incubation time. The present study reveals a strategy to realize in situ nanocomposites within LbL multilayer films simply by triggering the formation of protein fibrils by controlling the intermolecular interactions between amyloid precursors and polyelectrolytes (PEs).

Controlling and understanding the changes in metastatic cancer cell adhesion, shape, and motility are of paramount importance in cancer research, diagnosis, and treatment. Here, we used gold nanoparticles (AuNPs) as nanotopological structures and protein nanocluster forming substrates. Cell adhesion controlling proteins [in this case, fibronection (Fn) and ephrinB3] were modified to AuNPs, and these particles were then modified to the layer-by-layer (LbL) polymer surface that offers a handle for tuning surface charge and mechanical property of a cell-interfacing substrate. We found that metastatic cancer cell adhesion is affected by nanoparticle density on a surface, and ∼140 particles per 400 μm(2) (∼1.7 μm spacing between AuNPs) is optimal for effective metastatic cell adhesion. It was also shown that the AuNP surface density and protein nanoclustering on a spherical AuNP are controlling factors for the efficient interfacing and signaling of metastatic cancer cells. Importantly, the existence of nanotopological features (AuNPs in this case) is much more critical in inducing more dramatic changes in metastatic cell adhesion, protrusion, polarity, and motility than the presence of a cell adhesion protein, Fn, on the surface. Moreover, cell focal adhesion and motility-related paxillin clusters were heavily formed in cell lamellipodia and filopodia and high expression of phospho-paxillins were observed when the cells were cultured on either an AuNP or Fn-modified AuNP polymer surface. The ephrin signaling that results in the decreased expression of paxillin was found to be more effective when ephrins were modified to the AuNP surface than when ephrinB3 was directly attached to the polymer film. The overall trend for cell motility change is such that a nanoparticle-modified LbL surface induces higher cell motility and the AuNP modification to the LbL surface results in more pronounced change in cell motility than Fn or ephrin modification to the LbL surface.

We developed a cell-based assay based on the spin-assisted layer-by-layer (LbL) assembled polyelectrolyte matrix platforms. Three types of human breast epithelial cell lines including normal cells (184B5), noncancerous fibrocystic disease cells (MCF 10F), and metastatic cancerous cells (CAMA-1) were cultured, analyzed, and compared in parallel on various LbL-assembled polymer films. Poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) electrolyte polymers were used as the basic building units to form various LbL polyelectrolyte matrices. The mechanical rigidity, surface charge, and biorecognition property of the LbL platforms were controlled by tailoring the LbL surface, thermal cross-linking, and protein modification. Cellular phenotypic changes in adhesion, proliferation, and morphology on these LbL films were characterized and analyzed for the three different cell types. Our analysis results indicate that the cellular phenotype can be controlled by taking advantage of different surface charge, mechanical property, and biological modification (i.e., fibronectin in this case) of the LbL multilayer platforms. Importantly, cell phenotypical quantification results show that the cell spreading area per cell and optical density are useful parameters in distinguishing metastatic cancer cells from normal or fibrocystic disease cells on these LbL films. These LbL-based cell assay platforms have a potential for the development of various disease diagnostic cell assays.