From 1D Rods to 3D Networks: A Biohybrid Topological Diversity Investigated by Asymmetrical Flow Field-Flow Fractionation

Biohybrid structures formed by noncovalent interaction between avidin as a bridging unit and biotinylated glycodendrimers based on poly­(propyleneimine) (GD-B) have potential for biomedical application. Therefore, an exact knowledge about molar mass, dispersity, size, shape, and molecular structure is required. Asymmetrical flow field-flow fractionation (AF4) was applied to separate pure and assembled macromolecules according to their diffusion coefficients. The complex biohybrid structures consist of single components (avidin, differently valent GD-B) and nanostructures. These nanostructures were systematically studied depending on the degree of biotinylation and ligand–receptor stoichiometry by AF4 in combination with dynamic and static light scattering detection. This enables the quantification of composition and calculation of molar masses and radii, which were used to analyze scaling properties and apparent density of the formed structures. These data are compared to hydrodynamic radii obtained by applying the retention theory to the AF4 data. It is shown that depending on their architecture the molecular shape of biohybrid structures is changed from rod-like to spherical toward network-like behavior

Effect of Connectivity on the Structure and the Liquid–Solid Transition of Dense Suspensions of Soft Colloids

Aqueous solutions of multiarm flower-like poly­(ethylene oxide) (PEO) were formed and connected to various degrees by self-assembly. The structure was rendered permanent by <i>in situ</i> UV-irradiation. Dense suspensions of these single and connected soft colloids were studied by static and dynamic light scattering and viscosity measurements. The concentration dependence of the osmotic compressibility, the dynamic correlation length, and the viscosity of single flowers was shown to be close to that of equivalent PEO star-like polymers demonstrating that the effect of forming loops on the interaction is small. It was found that the osmotic compressibility and the dynamic correlation length of dense suspensions are not influenced by the bridging. However, when flower polymers are connected into clusters, motion in dense suspensions needs to be collective over larger length scales. This causes a much stronger increase of the viscosity for dense suspensions of interpenetrated clusters compared to single-flower polymers

Sphere-Like Protein–Glycopolymer Nanostructures Tailored by Polyassociation

Key parameters allow a reproducible polyassociation between avidin and biotinylated glycopolymers in order to fabricate defined supramolecular nanostructures for future (bio)­medical and biotechnological applications. Thus, the polymerization efficiency of biotinylated glycopolymers in the fabrication of biohybrid structures (BHS) was investigated with regard to the influence of (i) the degree of biotinylation of the dendritic glycoarchitectures, (ii) two biotin linkers, (iii) the dendritic scaffold (perfectly branched vs hyperbranched), and (iv) the ligand–receptor stoichiometry. The adjustment of all these parameters opens the way to fabricate defined sizes of the final biohybrid structures as a multifunctional platform ready for their use in different applications. Various analytical techniques, including purification of BHS, were used to gain fundamental insights into the structural properties of the resulting protein–glycopolymer BHS. Finally, the elucidation of pivotal conformational properties of isolated BHS with defined sizes by asymmetrical flow field flow fractionation study revealed that they mainly possess spherical-/star-like properties. From this study, the fundamental knowledge can be likely transferred to other assemblies formed by molecular recognition processes (e.g., adamantane-β-cyclodextrin)

Coil-like Enzymatic Biohybrid Structures Fabricated by Rational Design: Controlling Size and Enzyme Activity over Sequential Nanoparticle Bioconjugation and Filtration Steps

Well-defined enzymatic biohybrid structures (BHS) composed of avidin, biotinylated poly­(propyleneimine) glycodendrimers, and biotinylated horseradish peroxidase were fabricated by a sequential polyassociation reaction to adopt directed enzyme prodrug therapy to protein–glycopolymer BHS for potential biomedical applications. To tailor and gain fundamental insight into pivotal properties such as size and molar mass of these BHS, the dependence on the fabrication sequence was probed and thoroughly investigated by several complementary methods (e.g., UV/vis, DLS, cryoTEM, AF4-LS). Subsequent purification by hollow fiber filtration allowed us to obtain highly pure and well-defined BHS. Overall, by rational design and control of preparation parameters, e.g., fabrication sequence, ligand–receptor stoichiometry, and degree of biotinylation, well-defined BHS with stable and even strongly enhanced enzymatic activities can be achieved. Open coil-like structures of BHS with few branches are available by the sequential bioconjugation approach between synthetic and biological macromolecules possessing similar size dimensions

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction

In recent years, there has been growing attention to designing synthetic protocells, capable of mimicking micrometric and multicompartmental structures and highly complex physicochemical and biological processes with spatiotemporal control. Controlling metabolism-like cascade reactions in coacervate protocells is still challenging since signal transduction has to be involved in sequential and parallelized actions mediated by a pH change. Herein, we report the hierarchical construction of membraneless and multicompartmentalized protocells composed of (i) a cytosol-like scaffold based on complex coacervate droplets stable under flow conditions, (ii) enzyme-active artificial organelles and a substrate nanoreservoir capable of triggering a cascade reaction between them in response to a pH increase, and (iii) a signal transduction component based on the urease enzyme capable of the conversion of an exogenous biological fuel (urea) into an endogenous signal (ammonia and pH increase). Overall, this strategy allows a synergistic communication between their components within the membraneless and multicompartment protocells and, thus, metabolism-like enzymatic cascade reactions. This signal communication is transmitted through a scaffold protocell from an “inactive state” (nonfluorescent protocell) to an “active state” (fluorescent protocell capable of consuming stored metabolites)

Feedback-Induced and Oscillating pH Regulation of a Binary Enzyme–Polymersomes System

The stimuli-triggered regulating ability is a basic characteristic of biological systems, and it is always cyclic in nature. To mimic this stimuli-triggered regulation process for the construction of artificial cellular structures and functions is a challenge. Here, we present the development of artificial organelles system (AOS) with stimuli-trigged regulation ability consisting of the coexisting glucose oxidase-(GOx)-loaded pH-responsive polymersomes A (GOx-Psomes A) and urease-loaded pH-responsive polymersomes B (Urease-Psomes B) with orthogonal-responsive membranes. The addition of chemical fuels triggers the out-of-equilibrium state of AOS at which the pH decreases (glucose as fuel) or increases (urea as fuel). The pH change results in the catalytic “on” or “off” switch of GOx-Psomes A and Urease-Psomes B at different states due to their different pH responsiveness. Thus, this AOS with feedback-induced and oscillating pH-regulating ability paves the way for the construction of artificial eukaryotic cell biomimetics with controlled communications and metabolism mimicking

Redox- and pH-Responsive Polymersomes with Ferrocene Moieties Exhibiting Peroxidase-like, Chemoenzymatic Activity and H₂O₂‑Responsive Release Behavior

The development of compartments for the design of cascade reactions in a local space requires a selective spatiotemporal control. The combination of enzyme-loaded polymersomes with enzymelike units shows a great potential in further refining the diffusion barrier and the type of reactions in nanoreactors. Herein, pH-responsive and ferrocene-containing block copolymers were synthesized to realize pH-stable and multiresponsive polymersomes. Permeable membrane, peroxidase-like behavior induced by the redox-responsive ferrocene moieties and release properties were validated using cyclovoltammetry, dye TMB assay, and rupture of host–guest interactions with β-cyclodextrin, respectively. Due to the incorporation of different block copolymers, the membrane permeability of glucose oxidase-loaded polymersomes was changed by increasing extracellular glucose concentration and in TMB assay, allowing for the chemoenzymatic cascade reaction. This study presents a potent synthetic, multiresponsive nanoreactor platform with tunable (e.g., redox-responsive) membrane properties for potential application in therapeutics

Advancing Antiamyloidogenic Activity by Fine-Tuning Macromolecular Topology

Amyloid β peptide can aggregate into thin β-sheet fibrils or plaques deposited on the extracellular matrix, which is the hallmark of Alzheimer’s disease. Multifunctional macromolecular structures play an important role in inhibiting the aggregate formation of amyloidogenic materials and thus are promising candidates with antiamyloidogenic characteristics for the development of next-generation therapeutics. In this study, we evaluate how small differences in the dendritic topology of these structures influence their antiamyloidogenic activity by the comparison of “perfectly dendritic” and “pseudodendritic” macromolecules, both decorated with mannose units. Their compactness, the position of surface units, and the size of glyco-architectures influence their antiamyloidogenic activity against Aβ 40, a major component of amyloid plaques. For the advanced analysis of the aggregation of the Aβ peptide, we introduce asymmetric flow field flow fractionation as a suitable method for the quantification of large and delicate structures. This alternative method focuses on the quantification of complex aggregates of Aβ 40 and glycodendrimer/glyco-pseudodendrimer over different time intervals of incubation, showing a good correlation to ThT assay and CD spectroscopy results. Kinetic studies of the second-generation glyco-pseudodendrimer revealed maximum inhibition of Aβ 40 aggregates, verified with atomic force microscopy. The second-generation glyco-pseudodendrimer shows the best antiamyloidogenic properties confirming that macromolecular conformation in combination with optimal functional group distribution is the key to its performance. These molecular properties were validated and confirmed by molecular dynamics simulation

Bivalent Peptide- and Chelator-Containing Bioconjugates as Toolbox Components for Personalized Nanomedicine

While personalized therapy bears an enormous potential in cancer therapy, the development of flexible, tailorable delivery systems remains challenging. Here, we present a “tool-kit” of various avidin-based bioconjugates (BCs) for the preparation of personalized delivery systems. Corresponding BCs were synthesized using the self-assembly of avidin with various biotinylated ligands, such as one cationic glycodendrimer for dendriplex adsorption and two functional ligands for imaging (glycodendrimers with DOTA or NOTA units) or targeting (biotinylated PEG decorated with ligands). Substituting antibodies for targeting small molecules were coupled to biotin-PEG compounds for addressing the folate receptor (FR), epidermal growth factor receptor (EGFR), and prostate-specific membrane antigen (PSMA). After successful characterization and proof of good storage and redispersion properties of BCs, cytotoxicity assays and first in vivo imaging studies with 99mTc-complexing bioconjugates provide evidence that these BCs and their avidin analogues can be used as tool-kit components in theranostic systems for personalized medicine