Engineering exosome polymer hybrids by atom transfer radical polymerization.

Exosomes are emerging as ideal drug delivery vehicles due to their biological origin and ability to transfer cargo between cells. However, rapid clearance of exogenous exosomes from the circulation as well as aggregation of exosomes and shedding of surface proteins during storage limit their clinical translation. Here, we demonstrate highly controlled and reversible functionalization of exosome surfaces with well-defined polymers that modulate the exosome's physiochemical and pharmacokinetic properties. Using cholesterol-modified DNA tethers and complementary DNA block copolymers, exosome surfaces were engineered with different biocompatible polymers. Additionally, polymers were directly grafted from the exosome surface using biocompatible photo-mediated atom transfer radical polymerization (ATRP). These exosome polymer hybrids (EPHs) exhibited enhanced stability under various storage conditions and in the presence of proteolytic enzymes. Tuning of the polymer length and surface loading allowed precise control over exosome surface interactions, cellular uptake, and preserved bioactivity. EPHs show fourfold higher blood circulation time without altering tissue distribution profiles. Our results highlight the potential of precise nanoengineering of exosomes toward developing advanced drug and therapeutic delivery systems using modern ATRP methods.

Biomimetic Cell Structures: Probing Induced pH-Feedback Loops and pH Self-Monitoring in Cytosol Using Binary Enzyme-Loaded Polymersomes in Proteinosome.

Structures and functions of eukaryotic cells with an outer permeable membrane, a cytoskeleton, functional organelles, and motility can be mimicked by giant multicompartment protocells containing various synthetic organelles. Herein, two kinds of artificial organelles with stimuli-triggered regulation ability, glucose oxidase-(GOx)-loaded pH-responsive polymersomes A (GOx-Psomes A) and urease-loaded pH-responsive polymersomes B (Urease-Psomes B), and a pH-sensor (Dextran-FITC) are encapsulated into proteinosomes via the Pickering emulsion method. Thus, a polymersomes-in-proteinosome system is constructed which is able to probe biomimetic pH homeostasis. Alternating fuels (glucose or urea) introduced from outside the protocell penetrate the membrane of proteinosomes and enter into GOx-Psomes A and Urease-Psomes B to produce chemical signals (gluconic acid or ammonia) resulting in pH-feedback loops (pH jump and pH drop). This will counteract the catalytic "switch on" or "switch off" of enzyme-loaded Psomes A and B owing to their different pH-responsive membranes. Dextran-FITC in the proteinosome allows self-monitoring of slight pH fluctuations in the lumen of protocells. Overall, this approach shows heterogeneous polymersome-in-proteinosome architectures with sophisticated features such as input-regulated pH changes mediated by negative and positive feedback in loops and cytosolic pH self-monitoring, features that are imperative for advanced protocell design.

Advancing Antiamyloidogenic Activity by Fine-Tuning Macromolecular Topology.

Amyloid ß peptide can aggregate into thin ß-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 Aß 40, a major component of amyloid plaques. For the advanced analysis of the aggregation of the Aß 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 Aß 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 Aß 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.

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).

Crosslinked and Multi-Responsive Polymeric Vesicles as a Platform to Study Enzyme-Mediated Undocking Behavior: Toward Future Artificial Organelle Communication.

Various cellular functions are successfully mimicked, opening the door to the next generation of therapeutic approaches and systems biology. Herein, the first steps are taken toward the construction of artificial organelles for mimicking cell communication by docking and undocking of cargo in the membrane of swollen artificial organelles. Stimuli-responsive and crosslinked polymeric vesicles are used to allow docking processes at acidic pH at which ferrocene units in the swollen membrane state can undergo desired specific host-guest interaction using ß-cyclodextrin as model cargo. The release of the cargo mediated by two different enzymes, glucose oxidase and α-amylase, is investigated, triggered by distinct enzymatic undocking mechanisms. Different release times for a useful transport are shown that can be adapted to different communication pathways. In addition, Förster resonance energy transfer (FRET) experiments further support the hypotheses of host-guest inclusion complexation formation and their time-dependent breakdown. This work paves a way to a platform based on polymeric vesicles for synthetic biology, cell functions mimicking, and the construction of multifunctional cargo delivery system.

Probing Crowdedness of Artificial Organelles by Clustering Polymersomes for Spatially Controlled and pH-Triggered Enzymatic Reactions.

Most sophisticated biological functions and features of cells are based on self-organization, and the coordination and connection between their cell organelles determines their key functions. Therefore, spatially ordered and controllable self-assembly of polymersomes to construct clusters to simulate complex intracellular biological functions has attracted widespread attention. Here, we present a simple one-step copper-free click strategy to cross-link nanoscale pH-responsive and photo-cross-linked polymersomes (less than 100 nm) to micron-level clusters (more than 90% in 0.5-2 µm range). Various influencing factors in the clustering process and subsequent purification methods were studied to obtain optimal clustered polymeric vesicles. Even when polymeric vesicles separately loaded with different enzymes (glucose oxidase and myoglobin) are coclustered, the overall permeability of the clusters can still be regulated through tuning the pH values on demand. Compared with simple blending of those enzyme-loaded polymersomes, the rate of enzymatic cascade reaction increased significantly due to the interconnected complex microstructure established. The connection of catalytic nanocompartments into clusters confining different enzymes of a cascade reaction provides an excellent platform for the development of artificial systems mimicking natural organelles or cells.

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 ß-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.

Construction of Eukaryotic Cell Biomimetics: Hierarchical Polymersomes-in-Proteinosome Multicompartment with Enzymatic Reactions Modulated Protein Transportation.

The eukaryotic cell is a smart compartment containing an outer permeable membrane, a cytoskeleton, and functional organelles, presenting part structures for life. The integration of membrane-containing artificial organelles (=polymersomes) into a large microcompartment is a key step towards the establishment of exquisite cellular biomimetics with different membrane properties. Herein, an efficient way to construct a hierarchical multicompartment composed of a hydrogel-filled proteinosome hybrid structure with an outer homogeneous membrane, a smart cytoskeleton-like scaffold, and polymersomes is designed. Specially, this hybrid structure creates a micro-environment for pH-responsive polymersomes to execute a desired substance transport upon response to biological stimuli. Within the dynamic pH-stable skeleton of the protein hydrogels, polymersomes with loaded PEGylated insulin biomacromolecules demonstrate a pH-responsive reversible swelling-deswelling and a desirable, on-demand cargo release which is induced by the enzymatic oxidation of glucose to gluconic acid. This stimulus responsive behavior is realized by tunable on/off states through protonation of the polymersomes membrane under the enzymatic reaction of glucose oxidase, integrated in the skeleton of protein hydrogels. The integration of polymersomes-based hybrid structure into the proteinosome compartment and the stimuli-response on enzyme reactions fulfills the requirements of eukaryotic cell biomimetics in complex architectures and allows mimicking cellular transportation processes.

Molecular Dynamics-Guided Design of a Functional Protein-ATRP Conjugate That Eliminates Protein-Protein Interactions.

Even the most advanced protein-polymer conjugate therapeutics do not eliminate antibody-protein and receptor-protein recognition. Next-generation bioconjugate drugs will need to replace stochastic selection with rational design to select desirable levels of protein-protein interaction while retaining function. The "Holy Grail" for rational design would be to generate functional enzymes that are fully catalytic with small molecule substrates while eliminating interaction between the protein surface and larger molecules. Using chymotrypsin, an important enzyme that is used to treat pancreatic insufficiency, we have designed a series of molecular chimeras with varied grafting densities and shapes. Guided by molecular dynamic simulations and next-generation molecular chimera characterization with asymmetric flow field-flow fractionation chromatography, we grew linear, branched, and comb-shaped architectures from the surface of the protein by atom-transfer radical polymerization. Comb-shaped polymers, grafted from the surface of chymotrypsin, completely prevented enzyme inhibition with protein inhibitors without sacrificing the ability of the enzyme to catalyze the hydrolysis of a peptide substrate. Asymmetric flow field-flow fractionation coupled with multiangle laser light scattering including dynamic light scattering showed that nanoarmor designed with comb-shaped polymers was particularly compact and spherical. The polymer structure significantly increased protein stability and reduced protein-protein interactions. Atomistic molecular dynamic simulations predicted that a dense nanoarmor with long-armed comb-shaped polymer would act as an almost perfect molecular sieve to filter large ligands from substrates. Surprisingly, a conjugate that was composed of 99% polymer was needed before the elimination of protein-protein interactions.

Light-Driven Proton Transfer for Cyclic and Temporal Switching of Enzymatic Nanoreactors.

Temporal activation of biological processes by visible light and subsequent return to an inactive state in the absence of light is an essential characteristic of photoreceptor cells. Inspired by these phenomena, light-responsive materials are very attractive due to the high spatiotemporal control of light irradiation, with light being able to precisely orchestrate processes repeatedly over many cycles. Herein, it is reported that light-driven proton transfer triggered by a merocyanine-based photoacid can be used to modulate the permeability of pH-responsive polymersomes through cyclic, temporally controlled protonation and deprotonation of the polymersome membrane. The membranes can undergo repeated light-driven swelling-contraction cycles without losing functional effectiveness. When applied to enzyme loaded-nanoreactors, this membrane responsiveness is used for the reversible control of enzymatic reactions. This combination of the merocyanine-based photoacid and pH-switchable nanoreactors results in rapidly responding and versatile supramolecular systems successfully used to switch enzymatic reactions ON and OFF on demand.

Dehydropolymerisation of Methylamine Borane and an N-Substituted Primary Amine Borane Using a PNP Fe Catalyst.

Dehydropolymerisation of methylamine borane (H3 B⋅NMeH2 ) using the well-known iron amido complex [(PNP)Fe(H)(CO)] (PNP=N(CH2 CH2 PiPr2 )2 ) (1) gives poly(aminoborane)s by a chain-growth mechanism. In toluene, rapid dehydrogenation of H3 B⋅NMeH2 following first-order behaviour as a limiting case of a more general underlying Michaelis-Menten kinetics is observed, forming aminoborane H2 B=NMeH, which selectively couples to give high-molecular-weight poly(aminoborane)s (H2 BNMeH)n and only traces of borazine (HBNMe)3 by depolymerisation after full conversion. Based on a series of comparative experiments using structurally related Fe catalysts and dimethylamine borane (H3 B⋅NMe2 H) polymer formation is proposed to occur by nucleophilic chain growth as reported earlier computationally and experimentally. A silyl functionalised primary borane H3 B⋅N(CH2 SiMe3 )H2 was studied in homo- and co-dehydropolymerisation reactions to give the first examples for Si containing poly(aminoborane)s.

Avidin Localizations in pH-Responsive Polymersomes for Probing the Docking of Biotinylated (Macro)molecules in the Membrane and Lumen.

To mimic organelles and cells and to construct next-generation therapeutics, asymmetric functionalization and location of proteins for artificial vesicles is thoroughly needed to emphasize the complex interplay of biological units and systems through spatially separated and spatiotemporal controlled actions, release, and communications. For the challenge of vesicle (= polymersome) construction, the membrane permeability and the location of the cargo are important key characteristics that determine their potential applications. Herein, an in situ and post loading process of avidin in pH-responsive and photo-cross-linked polymersomes is developed and characterized. First, loading efficiency, main location (inside, lumen, outside), and release of avidin under different conditions have been validated, including the pH-stable presence of avidin in polymersomes' membrane outside and inside. This advantageous approach allows us to selectively functionalize the outer and inner membranes as well as the lumen with several bio(macro)molecules, generally suited for the construction of asymmetrically functionalized artificial organelles. In addition, a fluorescence resonance energy transfer (FRET) effect was used to study the permeability or uptake of the polymersome membrane against a broad range of biotinylated (macro)molecules (different typology, sizes, and shapes) under different conditions.

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.

Mono- and Polyassociation Processes of Pentavalent Biotinylated PEI Glycopolymers for the Fabrication of Biohybrid Structures with Targeting Properties.

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.

Effect of the Structure of Therapeutic Adenosine Analogues on Stability and Surface Electrostatic Potential of their Complexes with Poly(propyleneimine) Dendrimers.

Poly(propyleneimine) glycodendrimers are proposed as nanocarriers for triphosphate forms of anticancer adenosine analogues to improve the efficiency of chemotherapy and to overcome drug resistance mechanisms. This approach has proven successful for fludarabine administration-an autonomous way of cellular entry of a nucleotide-dendrimer noncovalent complex enables an increase in the intracellular accumulation and cytotoxic activity of the active metabolite of the drug. However, the attempt to apply an analogous strategy for clofarabine results in the inhibition of drug activity. To better understand this phenomenon, characterization and comparison of drug-dendrimer complexes were needed to indicate the differences in their surface properties and the strengths of fludarabine-dendrimer and clofarabine-dendrimer interactions. Here, zeta potential measurements, ultrafiltration, and asymmetric flow field-flow fractionation are applied to determine the surface electrostatic potential and stability of nucleotide-dendrimer formulations. This approach significantly extends the authors' research on the complexation potential of perfectly branched macromolecules, ultimately explaining previously observed differences and their consequences.

Tunable Fluorescence of a Semiconducting Polythiophene Positioned on DNA Origami.

A novel approach for the integration of π-conjugated polymers (CPs) into DNA-based nanostructures is presented. Using the controlled Kumada catalyst-transfer polycondensation, well-defined thiophene-based polymers with controllable molecular weight, specific end groups, and water-soluble oligoethylene glycol-based side chains were synthesized. The end groups were used for the easy but highly efficient click chemistry-based attachment of end-functionalized oligodeoxynucleotides (ODNs) with predesigned sequences. As demonstrated by surface plasmon resonance spectroscopy, the prepared block copolymers (BCPs), P3(EO)3T-b-ODN, comprising different ODN lengths and specific or repetitive sequences, undergo specific hybridization with complementary, thiol-functionalized ODNs immobilized on a gold surface. Furthermore, the site-specific attachment of the BCPs to DNA origami structures is studied. We demonstrate that a nanoscale object, that is, a single BCP with a single ODN handle, can be directed and bound to the DNA origami with reasonable yield, site-specificity, and high spatial density. On the basis of these results, we are able to demonstrate for the first time that optical properties of CP molecules densely immobilized on DNA origami can be locally fine-tuned by controlling the attractive π-π-stacking interactions between the CPs. In particular, we show that the fluorescence of the immobilized CP molecules can be significantly enhanced by surfactant-induced breakup of π-π-stacking interactions between the CP's backbones. Such molecular control over the emission intensity of the CPs can be valuable for the construction of sophisticated switchable nanophotonic devices and nanoscale biosensors.

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-ß-cyclodextrin).

Overcoming concealment effects of targeting moieties in the PEG corona: controlled permeable polymersomes decorated with folate-antennae for selective targeting of tumor cells.

In the context of diligent efforts to improve the tumor targeting efficiency of drug carriers, a shape-persistent polymersome which possess a pH-tunable membrane as well as folate targeting antennae is reported. The membrane of such polymersomes behaves as gate which undergoes "on" and "off" switches in response to pH stimuli. Thus, polymersomes can effectively prohibit the premature release of chemotherapeutic agents such as doxorubicin in physiological conditions, but promote drug release once they are triggered in the acidified endosomal compartment. Importantly, the folate moieties are installed on the surface of polymersomes as protruding antennae by doping the polymersomes with folate-terminated block copolymers designed to have longer PEG segments. Thereby, the folate moieties are freed from concealment and steric effects exerted by the dense PEG corona. The cellular uptake of the FA-antennae polymersomes by tumor cells is significantly enhanced facilitated by the freely accessible folate antennae; however, the normal cells record a low level of cellular uptake due to the stealth property of the PEG corona. Overall, the excellent biocompatibility, controlled permeability, targeted internalization, as well as selective cytotoxicity of such polymersomes set up the basis for properly smart carrier for targeted drug delivery.

Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Artificial Organelles with Digesting Characteristics: Imitating Simplified Lysosome- and Macrophage-Like Functions by Trypsin-Loaded Polymersomes.

Defects in cellular protein/enzyme encoding or even in organelles are responsible for many diseases. For instance, dysfunctional lysosome or macrophage activity results in the unwanted accumulation of biomolecules and pathogens implicated in autoimmune, neurodegenerative, and metabolic disorders. Enzyme replacement therapy (ERT) is a medical treatment that replaces an enzyme that is deficient or absent in the body but suffers from short lifetime of the enzymes. Here, this work proposes the fabrication of two different pH-responsive and crosslinked trypsin-loaded polymersomes as protecting enzyme carriers mimicking artificial organelles (AOs). They allow the enzymatic degradation of biomolecules to mimic simplified lysosomal function at acidic pH and macrophage functions at physiological pH. For optimal working of digesting AOs in different environments, pH and salt composition are considered the key parameters, since they define the permeability of the membrane of the polymersomes and the access of model pathogens to the loaded trypsin. Thus, this work demonstrates environmentally controlled biomolecule digestion by trypsin-loaded polymersomes also under simulated physiological fluids, allowing a prolonged therapeutic window due to protection of the enzyme in the AOs. This enables the application of AOs in the fields of biomimetic therapeutics, specifically in ERT for dysfunctional lysosomal diseases.