Glycooligomer-Functionalized Catalytic Nanocompartments Co-Loaded with Enzymes Support Parallel Reactions and Promote Cell Internalization.

A major shortcoming associated with the application of enzymes in drug synergism originates from the lack of site-specific, multifunctional nanomedicine. This study introduces catalytic nanocompartments (CNCs) made of a mixture of PDMS-b-PMOXA diblock copolymers, decorated with glycooligomer tethers comprising eight mannose-containing repeating units and coencapsulating two enzymes, providing multifunctionality by their in situ parallel reactions. Beta-glucuronidase (GUS) serves for local reactivation of the drug hymecromone, while glucose oxidase (GOx) induces cell starvation through glucose depletion and generation of the cytotoxic H2O2. The insertion of the pore-forming peptide, melittin, facilitates diffusion of substrates and products through the membranes. Increased cell-specific internalization of the CNCs results in a substantial decrease in HepG2 cell viability after 24 h, attributed to simultaneous production of hymecromone and H2O2. Such parallel enzymatic reactions taking place in nanocompartments pave the way to achieve efficient combinatorial cancer therapy by enabling localized drug production along with reactive oxygen species (ROS) elevation.

Synthetic molecular motor activates drug delivery from polymersomes.

The design of stimuli-responsive systems in nanomedicine arises from the challenges associated with the unsolved needs of current molecular drug delivery. Here, we present a delivery system with high spatiotemporal control and tunable release profiles. The design is based on the combination of an hydrophobic synthetic molecular rotary motor and a PDMS-b-PMOXA diblock copolymer to create a responsive self-assembled system. The successful incorporation and selective activation by low-power visible light (λ = 430 nm, 6.9 mW) allowed to trigger the delivery of a fluorescent dye with high efficiencies (up to 75%). Moreover, we proved the ability to turn on and off the responsive behavior on demand over sequential cycles. Low concentrations of photoresponsive units (down to 1 mol% of molecular motor) are shown to effectively promote release. Our system was also tested under relevant physiological conditions using a lung cancer cell line and the encapsulation of an Food and Drug Administration (FDA)-approved drug. Similar levels of cell viability are observed compared to the free given drug showing the potential of our platform to deliver functional drugs on request with high efficiency. This work provides an important step for the application of synthetic molecular machines in the next generation of smart delivery systems.

Trends in the Synthesis of Polymer Nano- and Microscale Materials for Bio-Related Applications.

Polymeric nano- and microscale materials bear significant potential in manifold applications related to biomedicine. This is owed not only to the large chemical diversity of the constituent polymers, but also to the various morphologies these materials can achieve, ranging from simple particles to intricate self-assembled structures. Modern synthetic polymer chemistry permits the tuning of many physicochemical parameters affecting the behavior of polymeric nano- and microscale materials in the biological context. In this Perspective, an overview of the synthetic principles underlying the modern preparation of these materials is provided, aiming to demonstrate how advances in and ingenious implementations of polymer chemistry fuel a range of applications, both present and prospective.

Amphiphilic Poly(vinyl alcohol) Membranes Leaving Out Chemical Cross-Linkers: Design, Synthesis, and Function of Tailor-Made Poly(vinyl alcohol)-b-poly(styrene) Copolymers.

Tailor-made poly(vinyl alcohol)-b-poly(styrene) copolymers (PVA-b-PS) for separation membranes are synthesized by the combination of reversible-deactivation radical polymerization techniques. The special features of these di-block copolymers are the high molecular weight (>70 kDa), the high PVA content (>80 wt%), and the good film-forming property. They are soluble only in hot dimethyl sulfoxide, but by the "solvent-switch" technique, they self-assemble in aqueous media to form micelles. When the self-assembled micelles are cast on a porous substrate, thin-film membranes with higher water permeance than that of PVA homopolymer are obtained. Thus, by using these tailor-made PVA-b-PS copolymers, it is demonstrated that chemical cross-linkers and acid catalysts can no longer be needed to produce PVA membranes, since the PS nanodomains within the PVA matrix act as cross-linking points. Lastly, subsequent thermal annealing of the thin film enhances the membrane selectivity due to the improved microphase separation.

Cell-Derived Vesicles with Increased Stability and On-Demand Functionality by Equipping Their Membrane with a Cross-Linkable Copolymer.

Cell-derived vesicles retain the cytoplasm and much of the native cell membrane composition. Therefore, they are attractive for investigations of membrane biophysics, drug delivery systems, and complex molecular factories. However, their fragility and aggregation limit their applications. Here, the mechanical properties and stability of giant plasma membrane vesicles (GPMVs) are enhanced by decorating them with a specifically designed diblock copolymer, cholesteryl-poly[2-aminoethyl methacrylate-b-poly(ethylene glycol) methyl ether acrylate]. When cross-linked, this polymer brush enhances the stability of the GPMVs. Furthermore, the pH-responsiveness of the copolymer layer allows for a controlled cargo loading/release, which may enable various bioapplications. Importantly, the cross-linked-copolymer GPMVs are not cytotoxic and preserve in vitro membrane integrity and functionality. This effective strategy to equip the cell-derived vesicles with stimuli-responsive cross-linkable copolymers is expected to open a new route to the stabilization of natural membrane systems and overcome barriers to biomedical applications.

Tailoring Polymer-Based Nanoassemblies for Stimuli-Responsive Theranostic Applications.

Polymer assemblies on the nanoscale represent a powerful toolbox for the design of theranostic systems when combined with both therapeutic compounds and diagnostic reporting ones. Here, recent advances in the design of theranostic systems for various diseases, containing-in their architecture-either polymers or polymer assemblies as one of the building blocks are presented. This review encompasses the general principles of polymer self-assembly, from the production of adequate copolymers up to supramolecular assemblies with theranostic functionality. Such polymer nanoassemblies can be further tailored through the incorporation of inorganic nanoparticles to endow them with multifunctional therapeutic and/or diagnostic features. Systems that change their architecture or properties in the presence of stimuli are selected, as responsivity to changes in the environment is a key factor for enhancing efficiency. Such theranostic systems are based on the intrinsic properties of copolymers or one of the other components. In addition, systems with a more complex architecture, such as multicompartments, are presented. Selected systems indicate the advantages of such theranostic approaches and provide a basis for further developments in the field.

Macroporous 3D Chitosan Cryogels for Fastac 10EC Pesticide Adsorption and Antibacterial Applications.

The pesticide pollution of surface water and wastewater has been recognized as a major worldwide concern due to their persistence in the aquatic environment and the potential adverse effects on human, flora, and fauna health. Apart from pesticides, bio-contamination with various bacterial populations leads to waterborne diseases. Hence, it becomes vital to remove the above-mentioned pollutants from water using a suitable process. Consequently, our study emphasized the potential benefits of a highly porous, chemically cross-linked 3D chitosan (CSGA) cryogel in the removal of pesticides and bacteria. The CSGA sponges were prepared using a facile and cost-effective approach that consisted of a three-step cryogenic process: (i) freezing at -18 °C, (ii) storage in a frozen state for a certain period, and (iii) thawing at room temperature. Batch adsorption experiments were performed under different environments, where the effects of several parameters, such as pH, contact time, and initial pollutant concentration were evaluated to identify the appropriate adsorption conditions for maximum pesticide removal. The CSGA-based cryogel sponges exhibited a theoretical maximum adsorption capacity of 160.82 mg g-1 for the Fastac 10EC pesticide and very good recyclability at room temperature. In addition, the antibacterial activities of these sponges were also investigated against various bacterial pathogens. The rates of killing Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus were close to 82%, 100%, and 99%, respectively. These results demonstrated that CSGA cryogels could be efficiently used in water remediation and find applications in the removal of pesticides and disinfection.

Inverting glucuronidation of hymecromone in situ by catalytic nanocompartments.

Glucuronidation is a metabolic pathway that inactivates many drugs including hymecromone. Adverse effects of glucuronide metabolites include a reduction of half-life circulation times and rapid elimination from the body. Herein, we developed synthetic catalytic nanocompartments able to cleave the glucuronide moiety from the metabolized form of hymecromone in order to convert it to the active drug. By shielding enzymes from their surroundings, catalytic nanocompartments favor prolonged activity and lower immunogenicity as key aspects to improve the therapeutic solution. The catalytic nanocompartments (CNCs) consist of self-assembled poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) diblock copolymer polymersomes encapsulating ß-glucuronidase. Insertion of melittin in the synthetic membrane of these polymersomes provided pores for the diffusion of the hydrophilic hymecromone-glucuronide conjugate to the compartment inside where the encapsulated ß-glucuronidase catalyzed its conversion to hymecromone. Our system successfully produced hymecromone from its glucuronide conjugate in both phosphate buffered solution and cell culture medium. CNCs were non-cytotoxic when incubated with HepG2 cells. After being taken up by cells, CNCs produced the drug in situ over 24 hours. Such catalytic platforms, which locally revert a drug metabolite into its active form, open new avenues in the design of therapeutics that aim at prolonging the residence time of a drug.

Membrane protein channels equipped with a cleavable linker for inducing catalysis inside nanocompartments.

Precisely timed initiation of reactions and stability of the catalysts are fundamental in catalysis. We introduce here an efficient closing-opening method for nanocompartments that contain sensitive catalysts and so achieve a controlled and extended catalytic activity. We developed a chemistry-oriented approach for modifying a pore-forming membrane protein which allows for a stimuli-responsive pore opening within the membrane of polymeric nanocompartments. We synthesized a diol-containing linker that selectively binds to the pores, blocking them completely. In the presence of an external stimulus (periodate), the linker is cleaved allowing the diffusion of substrate through the pores to the nanocompartment interior where it sets off the in situ enzymatic reaction. Besides the precise initiation of catalytic activity by opening of the pores, oxidation by periodate guarantees the cleavage of the linker under mild conditions. Accordingly, this kind of responsive nanocompartment lends itself to harboring a large variety of sensitive catalysts such as proteins and enzymes.

Physically cross-linked chitosan/dextrin cryogels entrapping Thymus vulgaris essential oil with enhanced mechanical, antioxidant and antifungal properties.

Herein, we entrapped Thymus vulgaris essential oil (EO) within the physically cross-linked sponge-like architecture of cryogels by ice template-assisted freeze-drying. Their 3D cryogenically-structured network was built through hydrogen bonding formed by blending two naturally-derived polysaccharides, chitosan and dextrin. The embedment of EOs within the cryogel matrix generates porous films with an increased elasticity that allows their fast shape recovery after full compression. Thus, the swollen EOs-loaded cryogel films exhibited an elastic modulus of 3.00 MPa, which is more than 40 times higher than that of polysaccharide films without EOs (an elastic modulus of only 0.07 MPa). In addition, the encapsulation of bioactive compounds endows the bio-based films with both antioxidant and antifungal properties, showing a radical scavenging activity of 65% and a zone inhibition diameter of 40 mm for Candida parapsilosis fungi. Our results recommend the entrapment of EOs into bio-based cryogel carriers as a straightforward approach to provide 'green' polysaccharide-based films having both improved physicochemical properties and remarkable antifungal activity.

Stabilizing Enzymes within Polymersomes by Coencapsulation of Trehalose.

Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzyme's original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature.

Design of Bio-Conjugated Hydrogels for Regenerative Medicine Applications: From Polymer Scaffold to Biomolecule Choice.

Bio-conjugated hydrogels merge the functionality of a synthetic network with the activity of a biomolecule, becoming thus an interesting class of materials for a variety of biomedical applications. This combination allows the fine tuning of their functionality and activity, whilst retaining biocompatibility, responsivity and displaying tunable chemical and mechanical properties. A complex scenario of molecular factors and conditions have to be taken into account to ensure the correct functionality of the bio-hydrogel as a scaffold or a delivery system, including the polymer backbone and biomolecule choice, polymerization conditions, architecture and biocompatibility. In this review, we present these key factors and conditions that have to match together to ensure the correct functionality of the bio-conjugated hydrogel. We then present recent examples of bio-conjugated hydrogel systems paving the way for regenerative medicine applications.

Composite cryo-beads of chitosan reinforced with natural zeolites with remarkable elasticity and switching on/off selectivity for heavy metal ions.

Combining ion-imprinting technology with pH-dependent adsorptive features of acid- or salt-activated zeolites brings up the opportunity to develop composite polymer materials with 'desired' sorption properties and performances. In this respect, we present here Co2+-imprinted composite cryo-beads with switching on/off selectivity towards the template ions, engineered by selecting the appropriate zeolite-treatment conditions and/or controlling the initial sorption pH values. Co2+ chelating efficiency of all cryo-beads was investigated either at pH 4 or 6 depending on zeolite conditioning strategy. The maximum sorption capacity values of ion-imprinted cryo-beads were from about 5 up to 7 times higher compared with those of non-imprinted ones. Under competitive conditions (Cu2+, Ni2+, Fe2+ and Cd2+ ions), the change of pH value from 4 to 6 resulted in a remarkable quenching of Co2+ selectivity generated by the zeolite shift from the H+-form to the Na+-form. The presence of zeolites within cryogel matrix generated composites with outstanding elasticity that allows the instant recovery of gels after full compression. These results indicate that the cryogel-type composites can be successfully re-used in separation processes for several times without losing their features.

The rise of bio-inspired polymer compartments responding to pathology-related signals.

Self-organized nano- and microscale polymer compartments such as polymersomes, giant unilamellar vesicles (GUVs), polyion complex vesicles (PICsomes) and layer-by-layer (LbL) capsules have increasing potential in many sensing applications. Besides modifying the physicochemical properties of the corresponding polymer building blocks, the versatility of these compartments can be markedly expanded by biomolecules that endow the nanomaterials with specific molecular and cellular functions. In this review, we focus on polymer-based compartments that preserve their structure, and highlight the key role they play in the field of medical diagnostics: first, the self-assembling abilities that result in preferred architectures are presented for a broad range of polymers. In the following, we describe different strategies for sensing disease-related signals (pH-change, reductive conditions, and presence of ions or biomolecules) by polymer compartments that exhibit stimuli-responsiveness. In particular, we distinguish between the stimulus-sensitivity contributed by the polymer itself or by additional compounds embedded in the compartments in different sensing systems. We then address necessary properties of sensing polymeric compartments, such as the enhancement of their stability and biocompatibility, or the targeting ability, that open up new perspectives for diagnostic applications.

Removal of heavy metal ions from multi-component aqueous solutions by eco-friendly and low-cost composite sorbents with anisotropic pores.

Copper, nickel, zinc, chromium, and iron ions are the prevailing contaminants in the aqueous effluents resulting from the photo-etching industry. In this context, we investigate here the metal ion sorption performance of an ion-imprinted cryogel (IIC), consisting of low-cost materials coming from renewable resources, towards multi-component metal ion solutions. The IIC sorbent, which is based on a chitosan matrix embedding a natural zeolite, was synthesized using a straightforward strategy by coupling copper-imprinting and unidirectional ice-templating methods. As consequence, the 1D-orientation and the interconnectivity of flow-channels sustain the fast metal ion diffusion within the IIC anisotropic structure. The removal efficiency of IIC sorbent reached 50% after 30 min, and the sorption equilibrium was attained within 150 min. For assessing the successful formation of imprinted cavities with well-defined sizes controlled by the radius of copper ions used as template, selectivity studies were performed on binary, ternary, and five-component synthetic mixtures. The efficiency of IIC as sorbent was further evaluated on real-life aqueous effluents discharged from photo-etching processes; thus, an IIC dosage of 6 g L-1 was found to remove 98.89% of Cu2+, 94.56% of Fe3+, 91.67% of Ni2+, 92.24% of Zn2+, and 82.76% of Cr3+ ions from this type of industrial wastewaters.

Could the porous chitosan-based composite materials have a chance to a "NEW LIFE" after Cu(II) ion binding?

Currently, biosorption is considered a leading-edge environmentally-friendly method for the low-cost remediation of wastewaters contaminated with metal ions. However, the safe disposal of metal-loaded biosorbents is still a challenging issue. In this context, our major objective was to explore the possibility of "waste minimization" by reusing the metal-loaded biosorbents in further environmental applications, particularly into the oxidative catalysis of dyes. Thus, the decolourisation efficiency (DE) of Methyl Orange (MO) in aqueous solutions under ambient light using copper-imprinted chitosan-based composites in comparison to non-imprinted ones was investigated in this work. The MO degradation was established first in the absence of any co-catalyst, when a DE value of 95.3% was achieved by the ion-imprinted catalysts within 360 min of reaction, compared to only 67.4% attained by the non-imprinted ones. Under Fenton-like conditions, the apparent degradation rate constant was seventy times higher, the DE increasing within 40 min to about 98.6%, and 70.5% respectively, whereas the content of co-catalyst (H2O2) was significantly lowered compared to other reported studies. The straightforward preparation of copper-loaded composites, along with their excellent stability and high efficiency even after four consecutive reaction runs support our ion-imprinted systems as potential catalysts for dye removal by oxidative decolourisation treatments.

Chitosan-based ion-imprinted cryo-composites with excellent selectivity for copper ions.

An original strategy is proposed here to design chitosan-based ion-imprinted cryo-composites (II-CCs) with pre-organized recognition sites and tailored porous structure by combining ion-imprinting and ice-templating techniques. The cryo-composites showed a tube-like porous morphology with interconnected parallel micro-channels, the distance between the channel walls being around 15 µm. Both the entrapment of a natural zeolite and the presence of carboxylate groups, generated by partial hydrolysis of amide moieties, led to II-CCs with controlled swelling ratios (25-40 g/g, depending on pH) and enhanced overall chelating efficiency (260 mg Cu2+/g composite). To point out the importance of introducing Cu2+ recognition sites, sorption experiments using mixtures of Cu2+ and other competing ions (Co2+, Ni2+, Zn2+ or/and Pb2+) were also carried out. The higher values of selectivity coefficients obtained for the II-CCs compared to those of non-imprinted ones highlight the remarkable potential of our sorbents for decontamination of wastewaters and recycling of Cu2+ ions.

Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes.

The development of advanced stimuli-responsive systems for medicine, catalysis, or technology requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to molecular transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein "gates" in the membrane. The outer membrane protein F (OmpF) porin was chemically modified with a pH-responsive molecular cap to serve as "gate" producing pH-driven molecular flow through the membrane and control the in situ enzymatic activity. This strategy provides complex reaction spaces necessary in "smart" medicine and for biomimetic engineering of artificial cells.

Does Membrane Thickness Affect the Transport of Selective Ions Mediated by Ionophores in Synthetic Membranes?

Biomimetic polymer nanocompartments (polymersomes) with preserved architecture and ion-selective membrane permeability represent cutting-edge mimics of cellular compartmentalization. Here it is studied whether the membrane thickness affects the functionality of ionophores in respect to the transport of Ca2+ ions in synthetic membranes of polymersomes, which are up to 2.6 times thicker than lipid membranes (5 nm). Selective permeability toward calcium ions is achieved by proper insertion of ionomycin, and demonstrated by using specific fluorescence markers encapsulated in their inner cavities. Preservation of polymersome architecture is shown by a combination of light scattering, transmission electron microscopy, and fluorescence spectroscopy. By using a combination of stopped-flow and fluorescence spectroscopy, it is shown that ionomycin can function and transport calcium ions across polymer membranes with thicknesses in the range 10.7-13.4 nm (7.1-8.9 times larger than the size of the ionophore). Thicker membranes induce a decrease in transport, but do not block it due to the intrinsic flexibility of these synthetic membranes. The design of ion selective biomimetic nanocompartments represents a new path toward the development of cellular ion nanosensors and nano-reactors, in which calcium sensitive biomacromolecules can be triggered for specific biological functions.

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 Ca(2+)-sensitive dye encapsulated inside the polymersome cavity prior to inserting the biopore. The calcium sensitive dye, ionomycin, and Ca(2+) 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.