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

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

Bionanoreactors: From Confined Reaction Spaces to Artificial Organelles

Inspired by natural compartments, polymer-based supramolecular assemblies (dendrimers, polymersomes, PICsomes, LBL capsules) have been engineered with a variety of sizes and properties to host biomolecules and thereby yield functional hybrid materials/systems. We present bionanoreactors that are designed by encapsulation/insertion of active compounds (proteins, enzymes, mimics) within the confined space of the assemblies, where these compounds are protected and act in situ. Accessibility to the inner space of the bionanoreactors is a key factor to enable catalytic reactions. For successful biomedical applications, a complex set of requirements regarding bionanoreactors is imposed. Relevant examples of nanoreactors used for the detection and treatment of pathologic conditions are provided, even though they are still in the early stage of research. An overview of this emerging nanoscience-based field and its potential for improved solutions in domains such as medicine are presented

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

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. (C) 2017 Elsevier Ltd. All rights reserved