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

Polymer-Lipid Hybrid Membranes as a Model Platform to Drive Membrane-Cytochrome c Interaction and Peroxidase-like Activity

Controllable attachment of proteins to material surfaces is very attractive for many applications including biosensors, bioengineered scaffolds or drug screening. Especially, redox proteins have received considerable attention as a model system not only to understand the mechanism of electron transfer in biological systems, but also the development of novel biosensors. However, current research attempts suffer from denaturation of the protein after its attachment to solid substrates. Here, we present how lipid, polymer and hybrid membranes based on mixtures of lipids and copolymers on a solid support provide a more favorable environment to drive selective and functional attachment of a model redox protein, cytochrome c (cyt c). Polymer membranes provided chemical versatility to support covalent attachment of cyt c, whereas lipid membranes provided flexibility and biocompatibility to support insertion of cyt c through its hydrophobic part. Hybrid membranes combine the most promising characteristics of both lipids and polymers and allowed attachment of cyt c with both covalent attachment and insertion driven by hydrophobic interactions. We then investigated the effect of different attachment strategies on the accessibility and peroxidase-like activity of cyt c, in the presence of different membranes. The real-time combination of cyt c with the planar membranes was investigated by quartz crystal microbalance with dissipation. It was possible to selectively drive the insertion of cyt c into a specific lipid domain of hybrid membranes. In addition, protein accessibility and its functionality were dependent on the specificity of the combination strategy: covalent conjugation of cyt c to polymer and hybrid membranes promoted higher accessibility and supported higher peroxidase-like activity. Taking together, the combination of biomolecules with planar membranes can be modulated in such a way to improve the accessibility of the biomolecules and their resulting functionality for the development of efficient âEuro�active surfacesâEuro�

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 peptideâEuro"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

Supported lipid bilayer platforms for investigating complement activation, termination and regulation

The complement system serves as the frontline of host defense for the human body against foreign entities, and is triggered via one or more of several signaling cascades that consist of interactions between soluble and cell surface-bound complement proteins. In this thesis, the objective is to explore how lipid membranes influence the initiation, amplification, termination and regulation of complement activation, utilizing supported lipid bilayer platforms as a model experimental system. The overall hypothesis of this work is that the supported lipid bilayer platform provides a useful model system to recapitulate key protein-lipid and protein-protein interactions involved in complement activation at lipid membrane interfaces, thereby establishing an experimental framework to decipher key trigger factors (e.g., membrane surface charge) as well as evaluate candidate inhibitors acting against complement convertases and membrane attack complex assemblies. Within this scope, four different experimental studies were conducted, leading to new insights into the mechanisms by which lipid membranes influence complement activation. In the first study, the influence of membrane surface change on the adsorption properties of different pattern recognition proteins involved in initiation of complement activation was investigated. In the second study, the effects of covalent and noncovalent tethering strategies on the self-assembly of the alternative pathway C3 convertase was monitored. It was identified that the C3b immobilization scheme is a critical factor governing convertase assembly, further enabling successful evaluation of a clinically relevant complement inhibitor, compstatin. In the third study, the assembly of the complement-terminating membrane attack complex was monitored onto supported lipid bilayer platforms, with particular attention to the influence of membrane surface charge. It was determined that the membrane attack complex assembly preferentially forms on negatively charged lipid membranes, indicating the importance of membrane electrostatics. In the fourth study, the inhibitory action of vitronectin and clusterin against the membrane attack complex assembly was evaluated, offering insight into the different mechanisms by which these two regulatory proteins inhibit complement activation. In summary, the findings presented in this thesis significantly contribute to the fundamental understanding of complement activation occurring at lipid membrane interfaces, and offer a new platform to characterize the mechanisms of action for a wide range of complement inhibitors including small molecules, antibodies, and peptides.Doctor of Philosophy (MSE

Probing the influence of tether density on tethered bilayer lipid membrane (tBLM)-peptide interactions

Tethered bilayer lipid membranes (tBLMs) represent a promising model membrane system that can host transmembrane proteins to serve as biosensors with exceptional detection performance. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we systematically investigated the influence of tether density on tBLM-peptide interactions and characterized the membrane binding dynamics of membrane-active peptides on tBLMs using AH peptide as a model. To achieve both physical stability and nanoscale separation from the support substrate, the tBLMs were fabricated on self-assembled monolayers (SAMs) obtained using a mixture of tether and spacer molecules with controlled tether-to-spacer ratio from 1:99 (T1) to 100:0 (T100). The solvent-assisted lipid bilayer (SALB) formation method was then employed to form the tBLMs, before the introduction of AH peptide. The QCM-D measurement responses indicated that the interactions between AH peptide and tBLMs involved peptide adsorption on the membrane layer followed by peptide translocation across the membrane. With increasing tether density, the abundance of hydrophobic groups within the tether chain led to stronger interactions and greater amount of translocated peptide. Depending on the tether density, this could result in significant structural transformation within the tBLM. Taken together, our work highlights the prospect of modulating membrane-peptide interactions by means of controlling the tBLM architecture, which will facilitate the creation of model membrane systems with highly tailored functionalities.Nanyang Technological UniversityNational Research Foundation (NRF)This work was supported by the National Research Foundation of Singapore through a Competitive Research Programme grant (NRF-CRP10-2012-07) and a Proof-of-Concept grant (NRF2015NRF-POC0001-19) as well as through the Center for Precision Biology at Nanyang Technological University. Minsub Chung was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2017018140)

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

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