PEO-b-PCL-b-PMOXA Triblock Copolymers: From Synthesis to Microscale Polymersomes with Asymmetric Membrane

We report a new family of amphiphilic ABC triblock copolymers: poly(ethylene oxide)-block-polycaprolactone-block-poly(2-methy1-2-oxazoline) (PEO-b-PCL-b-PMOXA). The synthesis is free of toxic reagents, well-controlled and results in polymers with D-M < 1.25 and PMOXA length up to 25 units (2 kDa). We compare the self assembly of PEO-b-PCL-b-PMOXA with PEO-b-PCL depending on PCL length and hydrophilic weight fraction (f) using the film rehydration method. Polymers self-assemble into different microscale structures, including polymersomes, which were studied by laser scanning microscopy. We proved the asymmetry of polymersome membrane by two independent methods, which confirmed the presence of a longer PEO block and the absence of a shorter PMOXA block on the outer surface of polymersomes

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

Tailoring a Solvent-Assisted Method for Solid-Supported Hybrid Lipid-Polymer Membranes

Combining amphiphilic block copolymers and phospholipids opens new opportunities for the preparation of artificial membranes. The chemical versatility and mechanical robustness of polymers together with the fluidity and biocompatibility of lipids afford hybrid membranes with unique properties that are of great interest in the field of bioengineering. Owing to its straightforwardness, the solvent-assisted method (SA) is particularly attractive for obtaining solid-supported membranes. While the SA method was first developed for lipids and very recently extended to amphiphilic block copolymers, its potential to develop hybrid membranes has not yet been explored. Here, we tailor the SA method to prepare solid-supported polymer-lipid hybrid membranes by combining a small library of amphiphilic diblock copolymers poly(dimethyl siloxane)-poly(2-methyl-2-oxazoline) and poly(butylene oxide)- block -poly(glycidol) with phospholipids commonly found in cell membranes including 1,2-dihexadecanoyl- sn -glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine, sphingomyelin, and 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(glutaryl). The optimization of the conditions under which the SA method was applied allowed for the formation of hybrid polymer-lipid solid-supported membranes. The real-time formation and morphology of these hybrid membranes were evaluated using a combination of quartz crystal microbalance and atomic force microscopy. Depending on the type of polymer-lipid combination, significant differences in membrane coverage, formation of domains, and quality of membranes were obtained. The use of the SA method for a rapid and controlled formation of solid-supported hybrid membranes provides the basis for developing customized artificial hybrid membranes

Biomimetic Planar Polymer Membranes Decorated with Enzymes as Functional Surfaces

Functional surfaces were generated by a combination of enzymes with polymer membranes composed of an amphiphilic, asymmetric block copolymer poly(ethyleneglycol)-block-poly(γ-methyl-ε-caprolactone)-block-poly[(2-dimethylamino)ethylmethacrylate]. First, polymer films formed at the air–water interface were transferred in different sequences onto silica solid support using the Langmuir–Blodgett technique, generating homogeneous monolayers and bilayers. A detailed characterization of these films provided insight into their properties (film thickness, wettability, topography, and roughness). On the basis of these findings, the most promising membranes were selected for enzyme attachment. Functional surfaces were then generated by the adsorption of two model enzymes that can convert phenol and its derivatives (laccase and tyrosinase), well known as high-risk pollutants of drinking and natural water. Both enzymes preserved their activity upon immobilization with respect to their substrates. Depending on the properties of the polymer films, different degrees of enzymatic activity were observed: bilayers provided the best conditions in terms of both overall stability and enzymatic activity. The interaction between amphiphilic triblock copolymer films and enzymes is exploited to engineer “active surfaces” with specific functionalities and high efficacy resulting from the intrinsic activity of the biomolecules that is preserved by an appropriate synthetic environment

Porphyrin-polymer nanocompartments: singlet oxygen generation and antimicrobial activity

A new water-soluble photocatalyst for singlet oxygen generation is presented. Its absorption extends to the red part of the spectrum, showing activity up to irradiation at 660 nm. Its efficiency has been compared to that of a commercial analogue (Rose Bengal) for the oxidation of L-methionine. The quantitative and selective oxidation was promising enough to encapsulate the photocatalyst in polymersomes. The singlet oxygen generated in this way can diffuse and remain active for the oxidation of L-methionine outside the polymeric compartment. These results made us consider the use of these polymersomes for antimicrobial applications. E. Coli colonies were subjected to oxidative stress using the photocatalyst-polymersome conjugates and nearly all the colonies were damaged upon extensive irradiation while under the same red LED light irradiation, liquid cultures in the absence of porphyrin or porphyrin-loaded polymersomes were unharme

Porphyrin Containing Polymersomes with Enhanced ROS Generation Efficiency: in vitro evaluation

Abstract Porphyrins are molecules possessing unique photophysical properties making them suitable for application in photodynamic therapy. The incorporation of porphyrins into natural or synthetic nano-assemblies such as polymersomes is a strategy to improve and prolong their therapeutic capacities and to overcome their limitations as therapeutic and diagnostic agents. Here, 5,10,15,20-tetrakis(1-(6-ethoxy-6-oxohexyl)-4-pyridin-1-io)-21H,23H-porphyrin tetrabromide porphyrin is inserted into polymersomes in order to demonstrate that the encapsulation enhances its ability to generate highly reactive singlet oxygen (1O2) upon irradiation in vitro. The photoactivation of the free and polymersome-encapsulated porphyrin is evaluated by electron spin resonance and cell viability assays on three different mammalian cell lines. The results indicate that by encapsulating the porphyrin, a controlled ROS delivery within the cells is achieved, at the same time avoiding side effects such as dark toxicity, non-specific porphyrin release and over time decreased activity in vitro. This work focuses on showing a not-toxic model system for modern therapeutic nanomedicine, which works under mild irradiation and dosage conditions

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

Effects of Silver Nanoparticles on Primary Mixed Neural Cell Cultures: Uptake, Oxidative Stress and Acute Calcium Responses

In the body, nanoparticles can be systemically distributed and then may affect secondary target organs, such as the central nervous system (CNS). Putative adverse effects on the CNS are rarely investigated to date. Here, we used a mixed primary cell model consisting mainly of neurons and astrocytes and a minor proportion of oligodendrocytes to analyze the effects of well-characterized 20 and 40 nm silver nanoparticles (SNP). Similar gold nanoparticles served as control and proved inert for all endpoints tested. SNP induced a strong size-dependent cytotoxicity. Additionally, in the low concentration range (up to 10 μg/ml of SNP), the further differentiated cultures were more sensitive to SNP treatment. For detailed studies, we used low/medium dose concentrations (up to 20 μg/ml) and found strong oxidative stress responses. Reactive oxygen species (ROS) were detected along with the formation of protein carbonyls and the induction of heme oxygenase-1. We observed an acute calcium response, which clearly preceded oxidative stress responses. ROS formation was reduced by antioxidants, whereas the calcium response could not be alleviated by antioxidants. Finally, we looked into the responses of neurons and astrocytes separately. Astrocytes were much more vulnerable to SNP treatment compared with neurons. Consistently, SNP were mainly taken up by astrocytes and not by neurons. Immunofluorescence studies of mixed cell cultures indicated stronger effects on astrocyte morphology. Altogether, we can demonstrate strong effects of SNP associated with calcium dysregulation and ROS formation in primary neural cells, which were detectable already at moderate dosage

Combinatorial Strategy for Studying Biochemical Pathways in Double Emulsion Templated Cell-Sized Compartments

Abstract Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi-step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/metabolites in synthetic cell-sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high-precision double-emulsion microfluidics that enable: i) tight control over the absolute and relative enzymatic contents inside the GUVs, reaching nearly 100% encapsulation and co-encapsulation efficiencies, and ii) functional reconstitution of biopores and membrane proteins in the GUVs polymeric membrane, thus supporting in situ reactions. GUVs equipped with biopores/membrane proteins and loaded with one or more enzymes are arranged in a variety of combinations that allow the study of a three-step cascade in multiple topologies. Due to the spatiotemporal control provided, optimum conditions for decreasing the accumulation of inhibitors are unveiled, and benefited from reactive intermediates to maximize the overall cascade efficiency in compartments. The non-system-specific feature of the novel strategy makes this system an ideal candidate for the development of new synthetic routes as well as for screening natural and more complex pathways