Planar Polymer Membranes Accommodate Functional Self-Assembly of Inserted Resorcinarene Nanocapsules.

Solid-supported polymer membranes (SSPMs) offer great potential in material and life sciences due to their increased mechanical stability and robustness compared to solid-supported lipid membranes. However, there is still a need for expanding the functionality of SSPMs by combining them with synthetic molecular assemblies. In this study, SSPMs served as a flexible matrix for the insertion of resorcinarene monomers and their self-assembly into functional hexameric resorcinarene capsules. Resorcinarene capsules provide a large cavity with affinity specifically for cationic and polyhydroxylated molecules. While the capsules are stable in apolar organic solvents, they disassemble when placed in polar solvents, which limits their application. Here, a solvent-assisted approach was used for copolymer membrane deposition on solid support and simultaneous insertion of the resorcinarene monomers. By investigation of the molecular factors and conditions supporting the codeposition of the copolymer and resorcinarene monomers, a stable hybrid membrane was formed. The hydrophobic domain of the membrane played a crucial role by providing a sufficiently thick and apolar layer, allowing for the self-assembly of the capsules. The capsules were functional inside the membranes by encapsulating cationic guests from the aqueous environment. The amount of resorcinarene capsules in the hybrid membranes was quantified by a combination of quartz-crystal microbalance with dissipation and liquid chromatography-mass spectrometry, while the membrane topography and layer composition were analyzed by atomic force microscopy and neutron reflectometry. Functional resorcinarene capsules inside SSPMs can serve as dynamic sensors and potentially as cross-membrane transporters, thus holding great promise for the development of smart surfaces.

Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2).

The mitotic kinesin-like protein 2 (MKlp2) plays a key role in the proper completion of cytokinetic abscission. Specifically, the C-terminal tail of MKlp2 (CTM peptides) offers a stable tethering on the plasma membrane and microtubule cytoskeleton in the midbody during abscission. However, little is known about the underlying mechanism of how the CTM peptides bind to the plasma membrane of the intercellular bridge. Herein, we identify the specific molecular interaction between the CTM peptides and phosphatidylinositol phosphate (PIP) receptors using quartz crystal microbalance-dissipation and atomic force microscopy force spectroscopic measurements. To systematically examine the effects of amino acids, we designed a series of synthetic 33-mer peptides derived from the wild-type (CTM1). First, we evaluated the peptide binding amount caused by electrostatic interactions based on 100% zwitterionic and 30% negatively charged model membranes, whereby the nonspecific attractions were nearly proportional to the net charge of peptides. Upon incubating with PIP-containing model membranes, the wild-type CTM1 and its truncated mutation showed significant PI(3)P-specific binding, which was evidenced by a 15-fold higher binding mass and 6-fold stronger adhesion force compared to other negatively charged membranes. The extent of the specific binding was predominantly dependent on the existence of S21, whereby substitution or deletion of S21 significantly hindered the binding affinity. Taken together, our findings based on a correlative measurement platform enabled the quantification of the nonelectrostatic, selective binding interactions of the C-terminal of MKlp2 to certain PIP receptors and contributed to understanding the molecular mechanisms on complete cytokinetic abscission in cells.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes.

Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications.

Design of Bio-Conjugated Hydrogels for Regenerative Medicine Applications: From Polymer Scaffold to Biomolecule Choice.

Bio-conjugated hydrogels merge the functionality of a synthetic network with the activity of a biomolecule, becoming thus an interesting class of materials for a variety of biomedical applications. This combination allows the fine tuning of their functionality and activity, whilst retaining biocompatibility, responsivity and displaying tunable chemical and mechanical properties. A complex scenario of molecular factors and conditions have to be taken into account to ensure the correct functionality of the bio-hydrogel as a scaffold or a delivery system, including the polymer backbone and biomolecule choice, polymerization conditions, architecture and biocompatibility. In this review, we present these key factors and conditions that have to match together to ensure the correct functionality of the bio-conjugated hydrogel. We then present recent examples of bio-conjugated hydrogel systems paving the way for regenerative medicine applications.

Recent Advances in Hybrid Biomimetic Polymer-Based Films: from Assembly to Applications.

Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Biomolecules Turn Self-Assembling Amphiphilic Block Co-polymer Platforms Into Biomimetic Interfaces.

Biological membranes constitute an interface between cells and their surroundings and form distinct compartments within the cell. They also host a variety of biomolecules that carry out vital functions including selective transport, signal transduction and cell-cell communication. Due to the vast complexity and versatility of the different membranes, there is a critical need for simplified and specific model membrane platforms to explore the behaviors of individual biomolecules while preserving their intrinsic function. Information obtained from model membrane platforms should make invaluable contributions to current and emerging technologies in biotechnology, nanotechnology and medicine. Amphiphilic block co-polymers are ideal building blocks to create model membrane platforms with enhanced stability and robustness. They form various supramolecular assemblies, ranging from three-dimensional structures (e.g., micelles, nanoparticles, or vesicles) in aqueous solution to planar polymer membranes on solid supports (e.g., polymer cushioned/tethered membranes,) and membrane-like polymer brushes. Furthermore, polymer micelles and polymersomes can also be immobilized on solid supports to take advantage of a wide range of surface sensitive analytical tools. In this review article, we focus on self-assembled amphiphilic block copolymer platforms that are hosting biomolecules. We present different strategies for harnessing polymer platforms with biomolecules either by integrating proteins or peptides into assemblies or by attaching proteins or DNA to their surface. We will discuss how to obtain synthetic structures on solid supports and their characterization using different surface sensitive analytical tools. Finally, we highlight present and future perspectives of polymer micelles and polymersomes for biomedical applications and those of solid-supported polymer membranes for biosensing.

Immobilization Strategies for Functional Complement Convertase Assembly at Lipid Membrane Interfaces.

The self-assembly formation of complement convertases-essential biomacromolecular complexes that amplify innate immune responses-is triggered by protein adsorption. Herein, a supported lipid bilayer platform was utilized to investigate the effects of covalent and noncovalent tethering strategies on the self-assembly of alternative pathway C3 convertase components, starting with C3b protein adsorption followed bythe addition of factors B and D. Quartz crystal microbalance-dissipation (QCM-D) experiments measured the real-time kinetics of convertase assembly onto supported lipid bilayers. The results demonstrate that the nature of C3b immobilization onto supported lipid bilayers is a key factor governing convertase assembly. The covalent attachment of C3b to maleimide-functionalized supported lipid bilayers promoted the self-assembly of functional C3 convertase in the membrane-associated state and further enabled successful evaluation of a clinically relevant complement inhibitor, compstatin. By contrast, noncovalent attachment of C3b to negatively charged supported lipid bilayers also permitted C3b protein uptake, albeit membrane-associated C3b did not support convertase assembly in this case. Taken together, the findings in this work demonstrate that the attachment scheme for immobilizing C3b protein at lipid membrane interfaces is critical for downstream C3 convertase assembly, thereby offering guidance for the design and evaluation of membrane-associated biomacromolecular complexes.

Quantitative Profiling of Nanoscale Liposome Deformation by a Localized Surface Plasmon Resonance Sensor.

Characterizing the shape of sub-100 nm, biological soft-matter particulates (e.g., liposomes and exosomes) adsorbed at a solid-liquid interface remains a challenging task. Here, we introduce a localized surface plasmon resonance (LSPR) sensing approach to quantitatively profile the deformation of nanoscale, fluid-phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes contacting a titanium dioxide substrate. Experimental and theoretical results validate that, due to its high sensitivity to the spatial proximity of phospholipid molecules near the sensor surface, the LSPR sensor can discriminate fine differences in the extent of ionic strength-modulated liposome deformation at both low and high surface coverages. By contrast, quartz crystal microbalance-dissipation (QCM-D) measurements performed with equivalent samples were qualitatively sensitive to liposome deformation only at saturation coverage. Control experiments with stiffer, gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes verified that the LSPR measurement discrimination arises from the extent of liposome deformation, while the QCM-D measurements yield a more complex response that is also sensitive to the motion of adsorbed liposomes and coupled solvent along with lateral interactions between liposomes. Collectively, our findings demonstrate the unique measurement capabilities of LSPR sensors in the area of biological surface science, including competitive advantages for probing the shape properties of adsorbed, nanoscale biological particulates.

Stealth Immune Properties of Graphene Oxide Enabled by Surface-Bound Complement Factor H.

With mounting evidence that nanomaterials can trigger adverse innate immune responses such as complement activation, there is increasing attention to the development of strategies that mask the complement-activating properties of nanomaterials. The current gold standard to reduce complement activation of nanomaterials is the covalent attachment of polymer coatings on nanomaterial surfaces, even though this strategy provides only moderate protection against complement activation. Akin to protein coronas that form on nanomaterial surfaces in physiological fluids, noncovalent strategies based on protein adsorption would offer a simplified, biomimetic approach to mitigate complement activation. Herein, we demonstrate that precoating graphene-based nanomaterials with purified, natural proteins enables regulatory control of nanomaterial-triggered complement activation. When the graphene-based nanomaterials were coated with complement factor H, nearly complete protection (>90% reduction) against complement activation (a "stealth effect") was achieved. By contrast, coating the nanomaterials with a passivating layer of bovine or human serum albumins achieved moderate protection (∼40% reduction), whereas immunoglobulin G amplified complement activation by several-fold. Taken together, our results demonstrate that surface-bound factor H, as well as serum albumins, can prevent graphene oxide-triggered complement activation, thereby offering a facile approach to inhibit complement activation completely down to naturally occurring levels.