Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles.

Self-organization of nanocomponents was mainly focused on solid nanoparticles, quantum dots, or liposomes to generate complex architectures with specific properties, but intrinsically limited or not developed enough, to mimic sophisticated structures with biological functions in cells. Here, we present a biomimetic strategy to self-organize synthetic nanocompartments (polymersomes) into clusters with controlled properties and topology by exploiting DNA hybridization to interconnect polymersomes. Molecular and external factors affecting the self-organization served to design clusters mimicking the connection of natural organelles: fine-tune of the distance between tethered polymersomes, different topologies, no fusion of clustered polymersomes, and no aggregation. Unexpected, extended DNA bridges that result from migration of the DNA strands inside the thick polymer membrane (about 12 nm) represent a key stability and control factor, not yet exploited for other synthetic nano-object networks. The replacement of the empty polymersomes with artificial organelles, already reported for single polymersome architecture, will provide an excellent platform for the development of artificial systems mimicking natural organelles or cells and represents a fundamental step in the engineering of molecular factories.

Strain-Promoted Thiol-Mediated Cellular Uptake of Giant Substrates: Liposomes and Polymersomes.

Simple cyclic disulfides under high tension mediate the uptake of giant substrates, that is, liposomes and polymersomes with diameters of up to 400 nm, into HeLa Kyoto cells. To place them at the surface of the vesicles, the strained disulfides were attached to the head-group of cationic amphiphiles. Bell-shaped dose response curves revealed self-activation of the strained amphiphiles by self-assembly into microdomains at low concentrations and self-inhibition by micelle formation at high concentrations. Poor colocalization of internalized vesicles with endosomes, lysosomes, and mitochondria indicate substantial release into the cytosol. The increasing activity with disulfide ring tension, inhibition with Ellman's reagent, and inactivity of maleimide and guanidinium controls outline a distinct mode of action that deserves further investigation and is promising for practical applications.

Interfacing Functional Systems.

The objective of molecular systems engineering is to move beyond functional components and primary systems, towards cumulate emergent properties in interfaced higher-order systems of unprecedented multifunctionality and sophistication.

Nanoscale engineering of low-fouling surfaces through polydopamine immobilisation of zwitterionic peptides.

We report a versatile approach for the design of substrate-independent low-fouling surfaces via mussel-inspired immobilisation of zwitterionic peptides. Using mussel-inspired polydopamine (PDA) coatings, zwitterionic glutamic acid- and lysine-based peptides were immobilised on various substrates, including noble metals, metal oxides, polymers, and semiconductors. The variation of surface chemistry and surface wettability upon surface treatment was monitored with X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Following peptide immobilisation, the surfaces became more hydrophilic due to the strong surface hydration compared with PDA-coated surfaces. The peptide-functionalised surfaces showed resistance to human blood serum adsorption and also effectively prevented the adhesion of gram-negative and gram-positive bacteria (i.e., Escherichia coli and Staphylococcus epidermidis) and mammalian cells (i.e., NIH 3T3 mouse embryonic fibroblast cells). The versatility of mussel-inspired chemistry combined with the unique biological nature and tunability of peptides allows for the design of low-fouling surfaces, making this a promising coating technique for various applications.

Direct bio-electrocatalysis of O2 reduction by Streptomyces coelicolor laccase orientated at promoter-modified graphite electrodes.

Bacterial laccase from Streptomyces coelicolor (SLAC) has been immobilised and orientated at promoter (pyrene and neocuproine)-modified electrodes productively both for direct electron transfer (ET) between the electrode and the T1 Cu site of SLAC and direct (unmediated) bio-electrocatalysis of dioxygen reduction. Its T1 Cu potential ranges between 471 and 318 mV versus the normal hydrogen electrode, at pH 5.5 and 8, respectively; this value is dependent both on the solution pH and electrode modification. In the presence of O2, Cu of the T2/T3 trinuclear centre is distinguished electrochemically at 748-623 mV. Depending on the promoter nature, different orientations of SLAC at pyrene- and neocuproine-modified electrodes can be followed from the kinetic analysis of the ET rates. Bio-electrocatalytic reduction of oxygen starts from the T1 Cu potentials of SLAC, and is most efficient at the promoter-modified electrodes, thereby demonstrating good performance both in neutral and basic media and in solutions with a high NaCl content, such as sea water. The obtained results allow consideration of a broader bioenergetic application of laccases as biocathodes operating directly in such environmental media as sea water and physiological fluids.

Combining polymers with the functionality of proteins: new concepts for atom transfer radical polymerization, nanoreactors and damage self-reporting materials.

Proteins are macromolecules with a great diversity of functions. By combining these biomolecules with polymers, exciting opportunities for new concepts in polymer sciences arise. This highlight exemplifies the aforementioned with current research results of our group. We review our discovery that the proteins horseradish peroxidase and hemoglobin possess ATRPase activity, i.e. they catalyze atom transfer radical polymerizations. Moreover, a permeabilization method for polymersomes is presented, where the photo-reaction of an α-hydroxyalkylphenone with block copolymer vesicles yields enzyme-containing nanoreactors. A further intriguing possibility to obtain functional nanoreactors is to enclose a polymerization catalyst into the thermosome, a protein cage from the family of chaperonins. Last but not least, fluorescent proteins are discussed as mechanoresponsive molecular sensors that report microdamages within fiber-reinforced composite materials.

Fluorescent protein senses and reports mechanical damage in glass-fiber-reinforced polymer composites.

Yellow fluorescent protein (YFP) is used as a mechanoresponsive layer at the fiber/resin interface in glass-fiber-reinforced composites. The protein loses its fluorescence when subjected to mechanical stress. Within the material, it reports interfacial shear debonding and barely visible impact damage by a transition from a fluorescent to a non-fluorescent state.