Mechanical Characterization of Hybrid Vesicles Based on Linear Poly(Dimethylsiloxane-b-Ethylene Oxide) and Poly(Butadiene-b-Ethylene Oxide) Block Copolymers

Poly(dimethylsiloxane-ethylene oxide) (PDMS-PEO) and poly(butadiene-b-ethylene oxide) (PBd-PEO) are two block copolymers which separately form vesicles with disparate membrane permeabilities and fluidities. Thus, hybrid vesicles formed from both PDMS-PEO and PBd-PEO may ultimately allow for systematic, application-specific tuning of vesicle membrane fluidity and permeability. However, given the relatively low strength previously noted for comb-type PDMS-PEO vesicles, the mechanical robustness of the resulting hybrid vesicles must first be confirmed. Toward this end, we have characterized the mechanical behavior of vesicles formed from mixtures of linear PDMS-PEO and linear PBd-PEO using micropipette aspiration. Tension versus strain plots of pure PDMS12-PEO46 vesicles revealed a non-linear response in the high tension regime, in contrast to the approximately linear response of pure PBd33-PEO20 vesicles. Remarkably, the area expansion modulus, critical tension, and cohesive energy density of PDMS12-PEO46 vesicles were each significantly greater than for PBd33-PEO20 vesicles, although critical strain was not significantly different between these vesicle types. PDMS12-PEO46/PBd33-PEO20 hybrid vesicles generally displayed graded responses in between that of the pure component vesicles. Thus, the PDMS12-PEO46/PBd33-PEO20 hybrid vesicles retained or exceeded the strength and toughness characteristic of pure PBd-PEO vesicles, indicating that future assessment of the membrane permeability and fluidity of these hybrid vesicles may be warranted

Current Perspectives on Synthetic Compartments for Biomedical Applications

Nano- and micrometer-sized compartments composed of synthetic polymers are designed to mimic spatial and temporal divisions found in nature. Self-assembly of polymers into compartments such as polymersomes, giant unilamellar vesicles (GUVs), layer-by-layer (LbL) capsules, capsosomes, or polyion complex vesicles (PICsomes) allows for the separation of defined environments from the exterior. These compartments can be further engineered through the incorporation of (bio)molecules within the lumen or into the membrane, while the membrane can be decorated with functional moieties to produce catalytic compartments with defined structures and functions. Nanometer-sized compartments are used for imaging, theranostic, and therapeutic applications as a more mechanically stable alternative to liposomes, and through the encapsulation of catalytic molecules, i.e., enzymes, catalytic compartments can localize and act in vivo. On the micrometer scale, such biohybrid systems are used to encapsulate model proteins and form multicompartmentalized structures through the combination of multiple compartments, reaching closer to the creation of artificial organelles and cells. Significant progress in therapeutic applications and modeling strategies has been achieved through both the creation of polymers with tailored properties and functionalizations and novel techniques for their assembly

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

Nanoreactors for local production and release of antibiotic

Implant infections are emerging as a grave medical problem.The number of medical and surgical procedures involving medical implant devices will continue to grow, for example due to aging of the population. Device-associated infections are a consequence of bacterial adhesion and subsequent biofilm formation at the implantation site. Due to the importance of this problem, intense research is being focused on finding new, efficient treatments. Conventional antibiotic therapies remain ineffective and very often lead to removal of the contaminated device. Various alternative strategies have been proposed, however, these suffer from many drawbacks. Tackling infections associated with medical implants remain a challenge. In this thesis, enzymatically active, covalently immobilized nanoreactors based on poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic block copolymer were designed and prepared. These nanoreactors catalyzed the conversion of prodrug molecules, which exhibit no antibacterial activity, to a drug active as an antibiotic. The enzymatic conversion was shown to occur only inside the nanoreactors. When these are immobilized they represent a novel, nanosized system whereby a drug will not be released to the entire body, but will be synthesized in situ. This strategy offers multiple advantages: long term production of antibacterial compounds due to the protection of the enzyme from proteolytic degradation, control of drug production at a specific rate for a specific period of time, and localized drug delivery. First, cationic ring opening polymerization was employed to synthesize the polymer. The self-assembly of this polymer was studied, as was the enzymatic activity of the resulting nanoreactor. The covalent attachment of the nanoreactors to a surface was realized by two different strategies: (i) attachment via an amino bond, involving Schiff base formation and its further reduction (ii) attachment via photo-cleavage by a phenyl azido linker. Both approaches resulted in successful, stable immobilization. The attached nanoreactors were characterized by surface-sensitive techniques such as scanning electron microscopy and atomic force microscopy. Experiments with bacteria were conducted to demonstrate the antimicrobial potential of surface immobilized enzymatically active nanoreactors. In summary, this thesis develops the concept of polymeric nanoreactors that synthesize drugs in situ to inhibit bacterial growth. Additionally, the immobilization methodologies elaborated within the scope of this work could be further adapted for potential applications in biotechnology and biosensing

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

Asymmetric amphiphilic triblock copolymers : synthesis, characterization and self-assembly

We developed a synthetic pathway to new amphiphilic ABC triblock copolymers with watersoluble blocks A and C and a hydrophobic middle block B. The synthesis involves a two-step polymerization. The prepolymer AB, constituted of poly(ethylene) oxide –b –poly(dimethyl) siloxane was prepared by anionic ring-opening polymerization of cyclic siloxanes, withandsiloxane units. The polymerization of strained cycles (e.g., D3) leads to polysiloxanes with monodisperse chains; the reaction time for anionic polymerization is lower and the yield of polymerization improved. Finally using the AB diblock copolymers as macroinitiators, a cationic polymerization of 2-methyloxazoline leads to asymmetric ABC triblock copolymers. As a model polymer we used an amphiphilic polyethylene oxide-b-polydimethylsiloxane-b-poly 2-methyloxazoline (PEO-b-PDMS-b-PMOXA) triblock copolymer. In aqueous solutions, this triblock copolymer self-assembles into well defined supramolecular aggregates. For certain compositions, the triblock copolymers form membrane like-superstructures and spherical vesicles in aqueous media. With the help of fluorescently labelled polymers, we were able to prove that the walls of these vesicles are asymmetric, due to the incompatibility between the hydrophilic chains: the blocks A and C are segregated on two different sides of the membrane. In case of nanometer-sized vesicles where membrane-curvature plays an important role we were even able to achieve a control over the membrane orientation, i.e., which of the two hydrophilic block is at the inner and which at the outer surface. This seems to be mainly gouverned by steric considerations: generally the smaller hydrophilic blocks are forced to the inner side where less space is available. Interestingly, the intrinsic asymmetry of the vesicular walls induced a directed insertion of transmembrane proteins. Using Aquaporinas a model system we employed immunoassay, immunofluorescence and immunogold labelling, to quantify the amount and the orientation of these proteins in the walls of the asymmetric ABC-block copolymer vesicles. The results clearly show a direct correlation between the membrane orientation and the prefered direction of the proteins. These studies indicate clearly that amphiphilic ABC triblock copolymers provide a convenient way to come to new materials with a directional functionality. Since they allow even a control over the orientation, they could allow to realize systems with a functionality that is reversed with respect to the biological model

Polymersomes Based Versatile Nanoplatforms for Controlled Drug Delivery and Imaging

Drug delivery systems made based on nanotechnology represent a novel drug carrier system that can change the face of therapeutics and diagnosis. Among all the available nanoforms polymersomes have wider applications due to their unique characteristic features like drug loading carriers for both hydrophilic and hydrophobic drugs, excellent biocompatibility, biodegradability, longer shelf life in the bloodstream and ease of surface modification by ligands. Polymersomes are defined as the artificial vesicles which are enclosed in a central aqueous cavity which are composed of self-assembly with a block of amphiphilic copolymer. Various techniques like film rehydration, direct hydration, nanoprecipitation, double emulsion technique and microfluidic technique are mostly used in formulating polymersomes employing different polymers like PEO-b-PLA, poly (fumaric/sebacic acid), poly(N-isopropylacrylamide) (PNIPAM), poly (dimethylsiloxane) (PDMS), and poly(butadiene) (PBD), PTMC-b-PGA (poly (dimethyl aminoethyl methacrylate)-b-poly(l-glutamic acid)) etc. Polymersomes have been extensively considered for the conveyance of therapeutic agents for diagnosis, targeting, treatment of cancer, diabetes etc. This review focuses on a comprehensive description of polymersomes with suitable case studies under the following headings: chemical structure, polymers used in the formulation, formulation methods, characterization methods and their application in the therapeutic, and medicinal filed

Lateral diffusion processes in biomimetic polymer membranes

Molecular self-assembly offers an important bottom-up approach to generate new materials with great potential for applications in nano-, life- and medical- sciences and engineering. The interest in “soft” materials suitable for the generation of artificial, biomimetic membranes has increased rapidly over the last years. These membranes combine the advantages of specificity and efficiency found in nature and the robustness and stability of synthetic materials from polymer science. There are currently two approaches to design biomimetic membranes. One uses natural phospholipids, while the other ones uses synthetic lipid mimics as the advanced alternative, which have shown great mechanical and chemical stability compared to their natural counterparts. This is important for technological application where durable devices are required. Biological membrane proteins, which provide selective and very efficient membrane transport, can be inserted into these synthetic block copolymer membranes. This combination of a synthetic membrane with biological membrane proteins is an intriguing phenomenon because the fundamental requirements for successful insertion are still matter of debate. One important issue is that polymeric membranes have thicknesses that exceed the height of the membrane proteins by several factors and the two lengths actually do not match. However, this significant height mismatch can be overcome by choosing a polymer with high flexibility, which has been shown to allow membrane proteins insertion in their active conformation. Flexibility and fluidity are essential membrane properties allowing successful generation of biomimetic membranes. In this thesis, the fluid properties of synthetic membranes composed of synthetic amphiphiles are studied based on a large library of block copolymers. These consist of poly(2-methyloxazoline) (PMOXA) and poly(dimethylsiloxane) (PDMS) and are used as diblock (PMOXA-b-PDMS, AB) and triblock (PMOXA-b-PDMS-b-PMOXA, ABA) copolymers. Variation of the molecular weight induces changes in the membrane thickness and thus the fluidity of the membrane. The diffusion of membrane proteins within synthetic triblock copolymer membranes was investigated. The study revealed that the membrane proteins are mobile even at hydrophobic mismatches of up to 7 nm, which is a factor of seven compared to mismatches existing in biological membranes. The advantage of PDMS-containing block copolymers is their enormous flexibility even at high molecular weights, which provides a similar membrane environment compared to biological phospholipid membranes. This explains and displays the ability of PDMS to compress in contact to membrane proteins. Their diffusion decreases steadily with increasing thickness mismatch. The importance of a very flexible polymer for the generation of biomimetic membranes was elucidated for membrane protein insertion, such as PDMS, which offers high fluidity and high membrane stability within membranes with even large thicknesses. The properties of these synthetic membranes investigated here, i.e. fluidity, lateral diffusion and membrane thickness, are important for the generation of biomimetic membranes for technological applications

A basis for molecular factories: multifunctionality and immobilization of biomolecule-polymer assemblies

Bio-inspired planar polymer membranes are synthetic membranes designed to be combined with biomolecules such as proteins, enzymes or peptides. These membranes provide both an increased mechanical stability as well as an environment to preserve the functionality of the biomolecules. In this thesis, two different kinds of planar membrane systems are demonstrated. In the first project, a sensor for phenolic compounds based on a bio-inspired polymer membrane was developed. Functional surfaces were generated by combining enzymes with polymer membranes composed of an amphiphilic, asymmetric block copolymer. Firstly, polymer films which were formed at the air-water interface were transferred onto silica solid support, by using the Langmuir-Blodgett method. The films were characterized according to their properties, including film thickness, wettability, topography, and roughness. The most promising membranes were used for enzyme attachment. Two model enzymes, laccase and tyrosinase, were adsorbed to the surface and their activity regarding the conversion of phenolic compounds was measured. This project is described in Chapter 1 in detail. In the second project, the interaction of the model pore-forming peptide melittin was studied in combination with a planar synthetic membrane. The investigation focused the interaction of melittin with amphiphilic block copolymer-based synthetic planar membranes as well as the insertion of melittin into these membranes to induce pore formation. Some specific molecular properties of the block copolymers and of the resulting membranes were selected for the investigation, such as hydrophilic to hydrophobic block ratio, membrane thickness and surface roughness. Through melittin addition to the synthetic membranes, melittin insertion requirements were better understood. This project is described in Chapter 2 in detail. Each chapter contains a separate introduction, material and methods section and conclusion and outlook specific to the project.20 In summary, in this thesis the properties of different combinations and applications of polymer-based membranes with biomolecules were investigated to a deeper level