Bio-hybrid polymer membranes as tools for mimicking cell compartments

In cells, membrane proteins naturally insert in lipid bilayers. The thickness of a lipid bilayer cell membrane is around 5 nm, with little variation in the hydrophobic mismatch (difference between the hydrophobic region of the membrane protein and the hydrophobic region of the spanned membrane) allowing them to function properly. In this work, the challenge was to identify the proper conditions in which selected ion channels (gramicidin), ion carriers (ionomycin), and other biopores (engineered α-hemolysins and glycerol facilitator) maintain their function in synthetic membranes of polymersomes with thicknesses up to 16 nm. This raised a set of questions. Gramicidin has a length of 2.5 nm, therefore: is it possible to insert and function in membranes up to 6 times thicker than the ion-channel’s size? Is there a limit of membrane thickness at which the inserted membrane protein does not function anymore? Does a biopore preserve its full known function in thicker membranes? How does an ionophore of 1.5 nm in diameter, such as ionomycin, move through a thick hydropobic layer of a polymerosme membrane? Is it possible to explain the mechanism of permeabilization in thick polymer membranes? Moreover, these biopores require solubilization in organic solvents or detergents which might also impact the permeabilization of the synthetic membranes. Is there a way of avoiding detergent/organic solvent induced permeabilization and thus preservation of the architecture of the vesicles? Is the permeability induced only by the successful insertion of biopores? The insertion of membrane proteins is just a part of the challenge, as the final 3D architecture of polymersomes might also be affected in presence of additional biomolecules. The system becomes even more complex once enzymes are involved, or the designed vesicular systems are attached on solid supports. Therefore, the list of questions can be extended. As a result, this thesis aims to answer many of the above listed questions. The proposed solutions, described in this body of work, represent the foundation for the development of nano-scaled biosensors, nanoreactors and active surfaces

Selective ion-permeable membranes by insertion of biopores into polymersomes

In nature there are various specific reactions for which highly selective detection or support is required to preserve their bio-specificity or/and functionality. In this respect, mimics of cell membranes and bio-compartments are essential for developing tailored applications in therapeutic diagnostics. Being inspired by nature, we present here biomimetic nanocompartments with ion-selective membrane permeability engineered by insertion of ionomycin into polymersomes with sizes less than 250 nm. As a marker to assess the proper insertion and functionality of ionomycin inside the synthetic membrane, we used a Ca2+-sensitive dye encapsulated inside the polymersome cavity prior to inserting the biopore. The calcium sensitive dye, ionomycin, and Ca2+ did not influence the architecture and the size of polymersomes. Successful ionomycin functionality inside the synthetic membrane with a thickness of 10.7 nm was established by a combination of fluorescence spectroscopy and stopped-flow spectroscopy. Polymersomes equipped with ion selective membranes are ideal candidates for the development of medical applications, such as cellular ion nanosensors or nanoreactors in which ion exchange is required to support in situ reactions

Artificial Organelles: Reactions inside Protein-Polymer Supramolecular Assemblies

Reactions inside confined compartments at the nanoscale represent an essential step in the development of complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approach of combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to create functional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality is provided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stability and robustness. The availability of a large variety of biomolecules and synthetic membranes allows the properties and functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systems as the basis for molecular factories

AQP1 Is Up-Regulated by Hypoxia and Leads to Increased Cell Water Permeability, Motility, and Migration in Neuroblastoma

The water channel aquaporin 1 (AQP1) has been implicated in tumor progression and metastasis. It is hypothesized that AQP1 expression can facilitate the transmembrane water transport leading to changes in cell structure that promote migration. Its impact in neuroblastoma has not been addressed so far. The objectives of this study have been to determine whether AQP1 expression in neuroblastoma is dependent on hypoxia, to demonstrate whether AQP1 is functionally relevant for migration, and to further define AQP1-dependent properties of the migrating cells. This was determined by investigating the reaction of neuroblastoma cell lines, particularly SH-SY5Y, Kelly, SH-EP Tet-21/N and SK-N-BE(2)-M17 to hypoxia, quantitating the AQP1-related water permeability by stopped-flow spectroscopy, and studying the migration-related properties of the cells in a modified transwell assay. We find that AQP1 expression in neuroblastoma cells is up-regulated by hypoxic conditions, and that increased AQP1 expression enabled the cells to form a phenotype which is associated with migratory properties and increased cell agility. This suggests that the hypoxic tumor microenvironment is the trigger for some tumor cells to transition to a migratory phenotype. We demonstrate that migrating tumor cell express elevated AQP1 levels and a hypoxic biochemical phenotype. Our experiments strongly suggest that elevated AQP1 might be a key driver in transitioning stable tumor cells to migrating tumor cells in a hypoxic microenvironment

Polymer capsules as micro-/nanoreactors for therapeutic applications: Current strategies to control membrane permeability

Polymer capsules, fabricated either with the aid of a sacrificial template or via the self-assembly of block copolymers into polymer vesicles (polymersomes), have attracted a great deal of attention for their potential use as micro-/nanoreactors and artificial organelles for therapeutic applications. Compared to other biomedical applications of polymer capsules, such as drug delivery vehicles, where the polymer shell undergoes irreversible disruption/rupture that allows the release of the payload, the polymer shell in polymer micro-/nanoreactors has to maintain mechanical integrity while allowing the selective diffusion of reagents/reaction products. In the present review, strategies that permit precise control of the permeability of the polymer shell while preserving its architecture are documented and critiqued. Together with these strategies, specific examples where these polymer capsules have been employed as micro-/nanoreactors as well as approaches to scale-up and optimize these systems along with future perspectives for therapeutic applications in several degenerative diseases are elucidated.This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) and is co-funded under the European Regional Development under Grant Number 13/RC/2073. A.L. and J.R.S. would like to acknowledge Basque Government (Department of Education, Language Policy and Culture) for a postdoctoral grant and project GIC IT-632-13 respectively. Spanish Ministry of Industry and Competitiveness for project MAT 2013-45559-P is also acknowledged. M.L. and C.P. gratefully acknowledge the financial support provided by the Swiss National Science Foundation (SNSF). A.P would like to acknowledge the European Cooperation in Science and Technology (COST) Action iPROMEDAI project (TD 1305)

Bio-catalytic nanocompartments for in situ production of glucose-6-phosphate

Cells are sophisticated biocatalytic systems driving a complex network of biochemical reactions. A bioinspired strategy to create advanced functional systems is to design confined spaces for complex enzymatic reactions by using a combination of synthetic polymer assemblies and natural cell components. Here, we developed bio-catalytic nanocompartments that contain phosphoglucomutase protected by a biomimetic polymer membrane, which was permeabilized for reactants through insertion of an engineered α-hemolysin pore protein. These bio-catalytic nanocompartments serve for production of glucose-6-phosphate, and thus possess great potential for applications in an incomplete glycolysis, pentose phosphate pathway, or in plant biological reactions

Antioxidant functionalized polymer capsules to prevent oxidative stress

Polymeric capsules exhibit significant potential for therapeutic applications as microreactors, where the bio-chemical reactions of interest are efficiently performed in a spatial and time defined manner due to the encapsulation of an active biomolecule (e.g., enzyme) and control over the transfer of reagents and products through the capsular membrane. In this work, catalase loaded polymer capsules functionalized with an external layer of tannic acid (TA) are fabricated via a layer-by-layer approach using calcium carbonate as a sacrificial template. The capsules functionalised with TA exhibit a higher scavenging capacity for hydrogen peroxide and hydroxyl radicals, suggesting that the external layer of TA shows intrinsic antioxidant properties, and represents a valid strategy to increase the overall antioxidant potential of the developed capsules. Additionally, the hydrogen peroxide scavenging capacity of the capsules is enhanced in the presence of the encapsulated catalase. The capsules prevent oxidative stress in an in vitro inflammation model of degenerative disc disease. Moreover, the expression of matrix metalloproteinase-3 (MMP-3), and disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5), which represents the major proteolytic enzymes in intervertebral disc, are attenuated in the presence of the polymer capsules. This platform technology exhibits potential to reduce oxidative stress, a key modulator in the pathology of a broad range of inflammatory diseases.This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Programme under Grant Number 13/RC/2073. A.L. and J.R.S. would like to acknowledge the Basque Government (Department of Education, Language Policy and Culture) for a postdoctoral grant and project GIC IT-632-13 respectively. The Spanish Ministry of Industry and Competitiveness for project MAT 2013-45559-P is also acknowledged. S.T., M.L. and C.P. gratefully acknowledge the financial support provided by the University of Basel and the Swiss National Science Foundation (SNSF). A.P. and J.R.S would like to acknowledge the European Cooperation in Science and Technology (COST) Action iPROMEDAI project (TD 1305)

Durable vesicles for reconstitution of membrane proteins in biotechnology

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with

Biomaterial based strategies to reconstruct the nigrostriatal pathway in organotypic slice co-cultures

Protection or repair of the nigrostriatal pathway represents a principal disease-modifying therapeutic strategy for Parkinson's disease (PD). Glial cell line-derived neurotrophic factor (GDNF) holds great therapeutic potential for PD, but its efficacious delivery remains difficult. The aim of this study was to evaluate the potential of different biomaterials (hydrogels, microspheres, cryogels and microcontact printed surfaces) for reconstructing the nigrostriatal pathway in organotypic co-culture of ventral mesencephalon and dorsal striatum. The biomaterials (either alone or loaded with GDNF) were locally applied onto the brain co-slices and fiber growth between the co-slices was evaluated after three weeks in culture based on staining for tyrosine hydroxylase (TH). Collagen hydrogels loaded with GDNF slightly promoted the TH+ nerve fiber growth towards the dorsal striatum, while GDNF loaded microspheres embedded within the hydrogels did not provide an improvement. Cryogels alone or loaded with GDNF also enhanced TH+ fiber growth. Lines of GDNF immobilized onto the membrane inserts via microcontact printing also significantly improved TH+ fiber growth. In conclusion, this study shows that various biomaterials and tissue engineering techniques can be employed to regenerate the nigrostriatal pathway in organotypic brain slices. This comparison of techniques highlights the relative merits of different technologies that researchers can use/develop for neuronal regeneration strategies

Polymersomes with engineered ion selective permeability as stimuli-responsive nanocompartments with preserved architecture

Following a biomimetic approach, we present here polymer vesicles (polymersomes) with ion selective permeability, achieved by inserting gramicidin (gA) biopores in their membrane. Encapsulation of pH-, Na+- and K+- sensitive dyes inside the polymersome cavity was used to assess the proper insertion and functionality of gA inside the synthetic membrane. A combination of light scattering, transmission electron microscopy, and fluorescence correlation spectroscopy was used to show that neither the size, nor the morphology of the polymersomes was affected by successful insertion of gA in the polymer membrane. Interestingly, proper insertion and functionality of gA were demonstrated for membranes with thicknesses in the range 9.2–12.1 nm, which are significantly greater than membrane lipid counterparts. Both polymersomes with sizes around 100 nm and giant unilamellar vesicles (GUVs) with inserted gA exhibited efficient time response to pH- and ions and therefore are ideal candidates for designing nanoreactors or biosensors for a variety of applications in which changes in the environment, such as variations of ionic concentration or pH, are required