Boosting Microfluidic Enzymatic Cascade Reactions with pH-Responsive Polymersomes by Spatio-Chemical Activity Control.

Microfluidic flow reactors permit the implementation of sensitive biocatalysts in polymeric environments (e.g., hydrogel dots), mimicking nature through the use of diverse microstructures within defined confinements. However, establishing complex hybrid structures to mimic biological processes and functions under continuous flow with optimal utilization of all components involved in the reaction process represents a significant scientific challenge. To achieve spatial, chemical, and temporal control for any microfluidic application, compartmentalization is required, as well as the unification of different sensitive compartments in the reaction chamber for the microfluidic flow design. This study presents a self-regulating microfluidic system fabricated by a sequential photostructuring process with an intermediate chemical process step to realize pH-sensitive hybrid structures for the fabrication of a microfluidic double chamber reactor for controlled enzymatic cascade reaction (ECR). The key point is the adaptation and retention of the function of pH-responsive horseradish peroxidase-loaded polymersomes in a microfluidic chip under continuous flow. ECR is successfully triggered and controlled by an interplay between glucose oxidase-converted glucose, the membrane state of pH-responsive polymersomes, and other parameters (e.g., flow rate and fluid composition). This study establishes a promising noninvasive regulatory platform for extended spatio-chemical control of current and future ECR and other cascade reaction systems.

Continuous Transformation from Membrane-less Coacervates to Membranized Coacervates and Giant Vesicles: toward Multicompartmental Protocells with Complex (Membrane) Architectures.

The membranization of membrane-less coacervates paves the way for the exploitation of complex protocells with regard to structural and cell-like functional behaviors. However, the controlled transformation from membranized coacervates to vesicles remains a challenge. This can provide stable (multi)phase and (multi)compartmental architectures through the reconfiguration of coacervate droplets in the presence of (bioactive) polymers, bio(macro)molecules and/or nanoobjects. Herein, we present a continuous protocell transformation from membrane-less coacervates to membranized coacervates and, ultimately, to giant hybrid vesicles. This transformation process is orchestrated by altering the balance of non-covalent interactions through varying concentrations of an anionic terpolymer, leading to dynamic processes such as spontaneous membranization of terpolymer nanoparticles at the coacervate surface, disassembly of the coacervate phase mediated by the excess anionic charge, and the redistribution of coacervate components in membrane. The diverse protocells during the transformation course provide distinct structural features and molecular permeability. Notably, the introduction of multiphase coacervates in this continuous transformation process signifies advancements toward the creation of synthetic cells with different diffusible compartments. Our findings emphasize the highly controlled continuous structural reorganization of coacervate protocells and represents a novel step toward the development of advanced and sophisticated synthetic protocells with more precise compositions and complex (membrane) structures.

Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

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).

Biomimetic Protocells Featuring Macrophage-Like Capture and Digestion of Protein Pathogens.

Modern medical research develops interest in sophisticated artificial nano- and microdevices for future treatment of human diseases related to biological dysfunctions. This covers the design of protocells capable of mimicking the structure and functionality of eukaryotic cells. The authors use artificial organelles based on trypsin-loaded pH-sensitive polymeric vesicles to provide macrophage-like digestive functions under physiological conditions. Herein, an artificial cell is established where digestive artificial organelles (nanosize) are integrated into a protocell (microsize). With this method, mimicking crossing of different biological barriers, capture of model protein pathogens, and compartmentalized digestive function are possible. This allows the integration of different components (e.g., dextran as stabilizing block) and the diffusion of pathogens in simulated cytosolic environment under physiological conditions. An integrated characterization approach is carried out, with identifying electrospray ionization mass spectrometry as an excellent detection method for the degradation of a small peptide such as ß-amyloid. The degradation of model enzymes is measured by enzyme activity assays. This work is an important contribution to effective biomimicry with the design of cell-like functions having potential for therapeutic action.

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.

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.

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.

Reversible Molecular Capture and Release in Microfluidics by Host-Guest Interactions in Hydrogel Microdots.

The integration of microscopic hydrogels with high specific surface area and physically reactive groups into microfluidic systems for selective molecular interactions is attracting increasing attention. Herein, the reversible capture and release of molecules through host-guest interactions of hydrogel dots in a microfluidic device is reported, which translates the supramolecular chemistry to the microscale conditions under continuous flow. Polyacrylamide (PAAm) hydrogel arrays with grafted ß-cyclodextrin (ß-CD)  modified poly(2-methyl-2-oxazoline) (CD-PMOXA) chains are fabricated by photopolymerization and integrated into a polydimethylsiloxane (PDMS)-on-glass chip. The ß-CD/adamantane (ß-CD/Ada) host-guest complex is confirmed by two dimensional Nuclear Overhauser Effect Spectroscopy NMR (2D NOESY NMR) prior to transfer to microfluidics. Ada-modified molecules are successfully captured by host-guest interaction formed between the CD-PMOXA grafted chains in the hydrogel network and the guest molecule in the solution. Furthermore, the captured molecules are released by perfusing free ß-CD with higher binding affinity than those grafted in the hydrogel array. A small guest molecule adamantane-fluorescein-isothiocyanate (Ada-FITC) and a macromolecular guest molecule (Ada-PMOXA-Cyanine 5 (Cy5)) are separately captured and released for three times with a release ratio up to 46% and 92%, respectively. The reproducible capture and release of functional molecules with different sizes demonstrates the stability of this hydrogel system in microfluidics and will provide an opportunity for future applications.

Protocells Capable of Generating a Cytoskeleton-like Structure from intracellular Membrane-active Artificial Organelles.

The intricate nature of eukaryotic cells with differently viscous intracellular compartments provides (membrane-active) enzymes to trigger time- and concentration-dependent processes in the intra-/extracellular matrix. Herein, we capitalize on membrane-active artificial organelles (AOs) to develop fluidic and stable proteinaceous membrane-based protocells. AOs in protocells induce the self-assembly of oligopeptides into an artificial cytoskeleton that underline their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane-active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane-active AOs in protocells thoroughly provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study demonstrates an important contribution to effective biomimicry of cell-like structures, shapes and functions.

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.

Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Assisted Reproduction.

Remotely controllable microrobots are appealing for various biomedical in vivo applications. In particular, in recent years, our group has focused on developing sperm-microcarriers to assist sperm cells with motion deficiencies or low sperm count (two of the most prominent male infertility problems) to reach the oocyte toward in-vivo-assisted fertilization. Different sperm carriers, considering their motion in realistic media and confined environments, have been optimized. However, the already-reported sperm carriers have been mainly designed to transport single sperm cell, with limited functionality. Thus, to take a step forward, here, the development of a 4D-printed multifunctional microcarrier containing soft and smart materials is reported. These microcarriers can not only transport and deliver multiple motile sperm cells, but also release heparin and mediate local enzymatic reactions by hyaluronidase-loaded polymersomes (HYAL-Psomes). These multifunctional facets enable in situ sperm capacitation/hyperactivation, and the local degradation of the cumulus complex that surrounds the oocyte, both to facilitate the sperm-oocyte interaction for the ultimate goal of in vivo assisted fertilization.

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.

Programmable synthetic cell networks regulated by tuneable reaction rates.

Coupled compartmentalised information processing and communication via molecular diffusion underpin network based population dynamics as observed in biological systems. Understanding how both compartmentalisation and communication can regulate information processes is key to rational design and control of compartmentalised reaction networks. Here, we integrate PEN DNA reactions into semi-permeable proteinosomes and characterise the effect of compartmentalisation on autocatalytic PEN DNA reactions. We observe unique behaviours in the compartmentalised systems which are not accessible under bulk conditions; for example, rates of reaction increase by an order of magnitude and reaction kinetics are more readily tuneable by enzyme concentrations in proteinosomes compared to buffer solution. We exploit these properties to regulate the reaction kinetics in two node compartmentalised reaction networks comprised of linear and autocatalytic reactions which we establish by bottom-up synthetic biology approaches.

Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers.

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise ß-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Synthesis and biological and physico-chemical characterization of glycodendrimers and oligopeptides for the treatment of systemic lupus erythematosus.

Anti-(ds)-DNA antibodies are the serological hallmark of Systemic Lupus Erythematosus (SLE). They assemble in the bloodstream with (ds)-DNA, forming immunocomplexes, which spread all over the body causing, among the other symptoms, lupic glomerulonephritis. Pathological manifestations of the disease may be reduced by destabilizing or inhibiting the formation of the immunocomplexes. In this respect, glycodendrimers showed peculiar interacting abilities towards this kind of biomolecule. Various generations of open-shell maltose-decorated poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) dendrimers and two oligopeptides with different polyethylene glycol units were synthesized and characterized, and then tested for their anti-SLE activity. The activity of glycodendrimers and oligopeptides was evaluated in human plasma from patients with SLE, compared to healthy plasma, by means of an enzyme-linked immunosorbent assay (ELISA), and electron paramagnetic resonance (EPR) characterization using spin-label and spin-probe techniques. Different strategies for the immunocomplex formation were tested. The results show that both kinds of glycodendrimers and oligopeptides inhibited the formation of immunocomplexes. Also, a partial breakdown of preformed immunocomplexes was observed. Both ELISA and EPR analyses indicated a better activity of glycodendrimers compared to oligopeptides, the 3rd generation PPI dendrimer being the most promising against SLE. This study highlights the possibility to develop a new class of dendritic therapeutics for the treatment of Lupus in pre-clinical studies.