Deepening the insight into poly(butylene oxide)-block-poly(glycidol) synthesis and self-assemblies: micelles, worms and vesicles

Aqueous self-assembly of amphiphilic block copolymers is studied extensively for biomedical applications like drug delivery and nanoreactors. The commonly used hydrophilic block poly(ethylene oxide) (PEO), however, suffers from several drawbacks. As a potent alternative, poly(glycidol) (PG) has gained increasing interest, benefiting from its easy synthesis, high biocompatibility and flexibility as well as enhanced functionality compared to PEO. In this study, we present a quick and well-controlled synthesis of poly(butylene oxide)- block -poly(glycidol) (PBO- b -PG) amphiphilic diblock copolymers together with a straight-forward self-assembly protocol. Depending on the hydrophilic mass fraction of the copolymer, nanoscopic micelles, worms and polymersomes were formed as well as microscopic giant unilamellar vesicles. The particles were analysed regarding their size and shape, using dynamic and static light scattering, TEM and Cryo-TEM imaging as well as confocal laser scanning microscopy. We have discovered a strong dependence of the formed morphology on the self-assembly method and show that only solvent exchange leads to the formation of homogenous phases. Thus, a variety of different structures can be obtained from a highly flexible copolymer, justifying a potential use in biomedical applications

Artificial Melanogenesis by Confining Melanin/Polydopamine Production inside Polymersomes

Melanin and polydopamine are potent biopolymers for the development of biomedical nanosystems. However, applications of melanin or polydopamine-based nanoparticles are limited by drawbacks related to a compromised colloidal stability over long time periods and associated cytotoxicity. To overcome these hurdles, a novel strategy is proposed that mimics the confinement of natural melanin in melanosomes. Melanosome mimics are developed by co-encapsulating the melanin/polydopamine precursors L-DOPA/dopamine with melanogenic enzyme Tyrosinase within polymersomes. The conditions of polymersome formation are optimized to obtain melanin/polydopamine polymerization within the cavity of the polymersomes. Similar to native melanosomes, polymersomes containing melanin/polydopamine show long-term colloidal stability, cell-compatibility, and potential for cell photoprotection. This novel kind of artificial melanogenesis is expected to inspire new applications of the confined melanin/polydopamine biopolymers

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�

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

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

Synthesis and Aqueous Self-assembly of Atactic and Isotactic Poly(butylene oxide)-block-poly(glycidol) Diblock Copolymers

Aqueous self-assembly of amphiphilic block copolymers (BCPs) is studied extensively for biomedical applications such as drug delivery, nano- or microreactors or artificial cell mimics. The commonly used poly(ethylene glycol) (PEG) as hydrophilic block and poly(dimethyl siloxane) (PDMS) as hydrophobic block suffer from several drawbacks regarding synthesis, reproducibility or biocompatibility. As potent alternatives, poly(glycidol) (PG) as hydrophilic block and poly(butylene oxide) (PBO) as hydrophobic block have gained increasing interest, benefiting from their easy synthesis, high biocompatibility and flexibility. In this thesis, a quick and well-controlled microwave-assisted synthesis of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG) amphiphilic BCPs is presented together with a straight-forward self-assembly protocol. Depending on the hydrophilic mass fraction of the BCPs, nanoscopic micelles, worms and polymersomes (Small Unilamellar Vesicles, SUVs) were formed as well as microscopic giant unilamellar vesicles (GUVs). The self-assemblies were analysed regarding their size and shape, using a combination of light scattering and electron and fluorescence microscopy techniques. A strong dependence of the formed morphology on the self-assembly method was discovered, proving that only solvent exchange led to the formation of homogenous phases. Additionally, this work takes advantage of the possibility to introduce chirality into the PBO-b-PG backbones to create fully isotactic BCPs. The commonly used isotactic BCPs such as poly(L-lactic acid) or poly(propylene oxide) typically exhibit (semi-) crystalline behaviour, inducing high membrane stiffness and limiting their applicability in systems involving membrane proteins or sensitive cargo. Here, isotactic yet fully amorphous PBO-b-PG BCPs are introduced in order to overcome these limitations. Three PBO-b-PG BCPs, differing solely in their tacticities (R/S, R and S), were synthesised and characterised regarding their structural, optical and thermal properties. Their self-assembly into homogenous phases of SUVs was analysed, revealing stability differences between SUVs composed of the different BCPs. Additionally, GUVs were prepared by double emulsion microfluidics. Only the atactic BCP formed GUVs which were stable over several hours, whereas GUVs composed of isotactic BCPs ruptured within several minutes after formation. The ability of atactic PBO-b-PG to form microreactors was elucidated by reconstituting the membrane protein OmpF in the GUVs membrane and performing an enzyme reaction inside its lumen. A comparison with the established PDMS-b-PMOXA revealed that PBO-b-PG GUVs were more permeable to hydrophilic substrates. Hence, this study sets the basis to create functional nano- or microreactors composed of fully amorphous isotactic BCPs. It allows to assess how BCP tacticity affects the formation, morphology, stability and membrane thickness of SUVs and GUVs without affecting the membrane flexibility. This, in turn, will open a path to access interaction of the membrane forming isotactic BCPs with chiral cargo or chiral membrane proteins

Catalytic polymersomes to produce strong and long-lasting bioluminescence

Here, we introduce an artificial bioluminescent nanocompartment based on the encapsulation of light-producing enzymes, luciferases, inside polymersomes. We exploit nanocompartmentalization to enhance luciferase stability in a cellular environment but also to positively modulate enzyme kinetics to achieve a long-lasting glow type signal. These features pave the way for expanding bioluminescence to nanotechnology-based applications

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles

Data underlying the figures in the publication “Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles”, published in Polym. Chem., 2021, 12, 5377-5389. https://pubs.rsc.org/en/content/articlelanding/2021/PY/D1PY00952D Table of contents: 1. Figure 1: Zip archive containing the experimental data (NMR spectra) for the kinetics of the PBO syntheses (Figure 1c) and the PBO-b-PEEGE syntheses (Figure 1d). 2. Figure 2: Zip archive containing the experimental data for Figure 2. Structural characterisation of the (S)-BCP (S)-PBO27-b-(S)-PG14 by 1H-NMR spectroscopy in methanol-d4. 3. Figure 3: Zip archive containing the experimental data for Figure 3. Chiral characterisation of the atactic and isotactic BCPs. 4. Figure 4: Zip archive containing the experimental data for Figure 4. TEM and Cryo-TEM characterisation of the SUVs formed by (a) (R/S)-PBO26-b-(R/S)-PG14, (b) (R)-PBO26-b-(R)-PG14 and (c) (S)-PBO27-b-(S)-PG14. 5. Figure 5: Zip archive containing the CLSM images for Figure 5. 6. Figure 6: Zip archive containing the images for Figure 6. Snapshots of the enzyme reaction within the cavity of (R/S)-BCP GUVs (b) with and (c) without reconstituted membrane protein OmpF, recorded by CLSM at different time points 7. Figure 7: Excel file containing the experimental data for Figure 7. Fluorescence intensity development of (a) (R/S)-BCP and (b) PDMS25-b-PMOXA10 GUVs resulting from the reaction of resorufin β-D-galactopyranoside (RGP) with β-galactosidase (β-Gal) to yield fluorescent resorufin. 8. Table 1: Zip archive containing the experimental data for Table 1. 9. Table 2: Zip archive containing the experimental data for Table 2

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Aqueous self-assembly of amphiphilic block copolymers is studied extensively for biomedical applications like drug delivery and nanoreactors. The commonly used hydrophilic block poly(ethylene oxide) (PEO), however, suffers from several drawbacks. As a potent alternative, poly(glycidol) (PG) has gained increasing interest, benefiting from its easy synthesis, high biocompatibility and flexibility as well as enhanced functionality compared to PEO. In this study, we present a quick and well-controlled synthesis of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG) amphiphilic diblock copolymers together with a straight-forward self-assembly protocol. Depending on the hydrophilic mass fraction of the copolymer, nanoscopic micelles, worms and polymersomes were formed as well as microscopic giant unilamellar vesicles. The particles were analysed regarding their size and shape, using dynamic and static light scattering, TEM and Cryo-TEM imaging as well as confocal laser scanning microscopy. We have discovered a strong dependence of the formed morphology on the self-assembly method and show that only solvent exchange leads to the formation of homogenous phases. Thus, a variety of different structures can be obtained from a highly flexible copolymer, justifying a potential use in biomedical applications. This journal is © The Royal Society of Chemistry