Investigation of the Thermal Stability of Proteinase K for the Melt Processing of Poly(l-lactide).

The enzymatic degradation of aliphatic polyesters offers unique opportunities for various use cases in materials science. Although evidently desirable, the implementation of enzymes in technical applications of polyesters is generally challenging due to the thermal lability of enzymes. To prospectively overcome this intrinsic limitation, we here explored the thermal stability of proteinase K at conditions applicable for polymer melt processing, given that this hydrolytic enzyme is well established for its ability to degrade poly(l-lactide) (PLLA). Using assorted spectroscopic methods and enzymatic assays, we investigated the effects of high temperatures on the structure and specific activity of proteinase K. Whereas in solution, irreversible unfolding occurred at temperatures above 75-80 °C, in the dry, bulk state, proteinase K withstood prolonged incubation at elevated temperatures. Unexpectedly little activity loss occurred during incubation at up to 130 °C, and intermediate levels of catalytic activity were preserved at up to 150 °C. The resistance of bulk proteinase K to thermal treatment was slightly enhanced by absorption into polyacrylamide (PAM) particles. Under these conditions, after 5 min at a temperature of 200 °C, which is required for the melt processing of PLLA, proteinase K was not completely denatured but retained around 2% enzymatic activity. Our findings reveal that the thermal processing of proteinase K in the dry state is principally feasible, but equally, they also identify needs and prospects for improvement. The experimental pipeline we establish for proteinase K analysis stands to benefit efforts directed to this end. More broadly, our work sheds light on enzymatically degradable polymers and the thermal processing of enzymes, which are of increasing economical and societal relevance.

Modulation of Transaminase Activity by Encapsulation in Temperature-Sensitive Poly(N-acryloyl glycinamide) Hydrogels.

Smart hydrogels hold much potential for biocatalysis, not only for the immobilization of enzymes, but also for the control of enzyme activity. We investigated upper critical solution temperature-type poly N-acryloyl glycinamide (pNAGA) hydrogels as a smart matrix for the amine transaminase from Bacillus megaterium (BmTA). Physical entrapment of BmTA in pNAGA hydrogels results in high immobilization efficiency (>89 %) and high activity (97 %). The temperature-sensitiveness of pNAGA is preserved upon immobilization of BmTA and shows a gradual deswelling upon temperature reduction. While enzyme activity is mainly controlled by temperature, deactivation tended to be higher for immobilized BmTA (≈62-68 %) than for free BmTA (≈44 %), suggesting a deactivating effect due to deswelling of the pNAGA gel. Although the deactivation in response to hydrogel deswelling is not yet suitable for controlling enzyme activity sufficiently, it is nevertheless a good starting point for further optimization.

Perfusion Cultivation of Artificial Liver Extracellular Matrix in Fibrous Polymer Sponges Biomimicking Scaffolds for Tissue Engineering.

A major challenge in tissue engineering and artificial scaffolding is to combine easily tunable scaffolds biomimicking the extracellular matrix of native organs with delivery-controlled cell culturing to create fully cellularized, large artificial 3D scaffolds. Aiming at bioartificial liver construction, we present our research using galactose-functionalized, ultraporous polylactide 3D nanofiber sponges fabricated out of electrospun fibers. Sponge biomodification by blend galactosylation and in-solution coating is performed, respectively, using a polylactide-galactose carrier-copolymer that promotes cell delivery and features a pronounced autofluorescence. It allows us to verify the galactosylation success, evaluate its quality, and record dye-free, high-resolution images of the sponge network using confocal laser scanning microscopy. The galactose carrier and its impact on scaffold cellularization is validated in benchmark to several reference systems. Verification of the human hepatic cell asialoglycoprotein receptor presence and galactose interaction in culture is performed by Cu2+ receptor-blocking experiments. The culture results are extensively investigated in and ex situ to trace and quantify the cell culture progress, cell activity, and viability at different culture stages. Bioreactor cultivation of sponges reveals that the galactose carrier does not only facilitate cell adhesion but also enhances cellular distribution throughout the scaffold. The promising 3D culture results allow us to move forward to create mature in vitro liver model research systems. The elaboration into ex vivo testing platforms could help judging native cell material interactions with drugs or therapeutics, without the need of direct human or animal testing.

UCST-Type Copolymer through the Combination of Water-Soluble Polyacrylamide and Polycaprolactone-Like Polyester.

Copolymers of acrylamide (AAm) and hydrophobic monomers are known as non-ionic polymers to show an upper critical solution temperature (UCST) in aqueous solution. By utilizing cyclic ketene acetals and carrying out the radical ring opening polymerization along with copolymerization with AAm, a UCST polymer, poly(acrylamide-co-2-methylene-1,3-dioxepane) (P(AAm-co-MDO)), is obtained. The AAm/MDO content results in the shift of UCST phase transition temperature. The present work shows how the combination of water-soluble PAAm and polycaprolactone-like structured PMDO develops a new type of thermoresponsive polymer which can be expected for bio-related applications.

Wolf-Lamb-type Catalysis in One Pot Using Electrospun Polymeric Catalyst Membranes.

Multistep catalytic transformations using incompatible catalysts (Wolf-Lamb-type) in a one-pot reaction cascade require site isolation of different catalysts by compartmentalization. In this work, the use of different electrospun catalytic membranes in a modular way as individual compartments is shown for one-pot Wolf-Lamb-type reaction cascades. The data are presented for one-pot cascade reaction sequences catalyzed by acidic and basic membranes made by electrospinning polymeric acid (poly(styrene-co-styrene sulfonic acid-co-4-methacryloyl-oxybenzophen)) and basic (poly(styrene-co-4-vinylpyridine-co-4-methacryloyl-oxybenzophen)) catalysts, respectively. The two-step, one-pot system used is the acidic catalyzed deacetylation of dimethoxybenzylacetale to benzaldehyde, which reacts with ethyl cyanoformate to result in a high yield of product (over 90%) under base-catalyzed conditions. The reaction kinetics are further monitored and evaluated by using differential equations, showing the necessity of a parameter Δt to represent a retarded start for the second reaction step. The concept provides an easy and upscalable approach for use in Wolf-Lamb-type systems.

Let There be Light: Polymeric Micelles with Upper Critical Solution Temperature as Light-Triggered Heat Nanogenerators for Combating Drug-Resistant Cancer.

Complete drug release and efficient drug retention are two critical factors in reversing drug resistance in cancer therapy. In this regard, polymeric micelles with an upper critical solution temperature (UCST) are designed as a new exploration to reverse drug resistance. The amphiphilic UCST-type block copolymers are used to encapsulate photothermal agent IR780 and doxorubicin (DOX) simultaneously. The integrated UCST-type drug nanocarriers show light-triggered multiple synergistic effects to reverse drug resistance and are expected to kill three birds with one stone: First, owing to the photothermal effect of IR780, the nanocarriers will be dissociated upon exposure to laser irradiation, leading to complete drug release. Second, the photothermal effect-induced hyperthermia is expected to avoid the efflux of DOX and realize efficient drug retention. Last but not least, photothermal ablation of cancer cells can be achieved after laser irradiation. Therefore, the UCST-type drug nanocarriers provide a new strategy in reversing drug resistance in cancer therapy.

Ultraporous, Compressible, Wettable Polylactide/Polycaprolactone Sponges for Tissue Engineering.

Ultraporous, degradable sponges made of either polylactide or of blends of polylactide/poly(ε-caprolactone) are prepared by freeze-drying of dispersions of short electrospun fibers and subsequent thermal annealing. The sponges feature ultrahigh porosity (99.6%), a hierarchical cellular structure, and high reversible compressibility with fast recovery from deformation in the dry as well as in the wet state. The sponge properties depend on the fiber dispersion concentration and the annealing temperature. Sponge characteristics like fiber density (2.5-20 mg/cm3), size, shape, crystallinity, mechanical strength, wetability, and structural integrity are user adjustable. Cell culture experiments were successfully performed with Jurkat cells with Confocal Laser Scanning Microscopy and MTT staining showing rapid cell proliferation. Live/Dead staining demonstrated high viability of the seeded cells. The sponge characteristics and modifications investigated and presented here reveal that these sponges are highly promising for tissue engineering applications.

pH-Responsive Biohybrid Carrier Material for Phenol Decontamination in Wastewater.

Smart polymers are a valuable platform to protect and control the activity of biological agents over a wide range of conditions, such as low pH, by proper encapsulation. Such conditions are present in olive oil mill wastewater with phenol as one of the most problematic constituents. We show that elastic and pH-responsive diblock copolymer fibers are a suitable carrier for Corynebacterium glutamicum, i.e., bacteria which are known for their ability to degrade phenol. Free C. glutamicum does not survive low pH conditions and fails to degrade phenol at low pH conditions. Our tea-bag like biohybrid system, where the pH-responsive diblock copolymer acts as a protecting outer shell for the embedded bacteria, allows phenol degradation even at low pH. Utilizing a two-step encapsulation process, planktonic cells were first encapsulated in poly(vinyl alcohol) to protect the bacteria against the organic solvents used in the second step employing coaxial electrospinning.

Tuning the Phase Transition from UCST-Type to LCST-Type by Composition Variation of Polymethacrylamide Polymers.

Polymethacrylamide copolymers with hydrophobic N-substituted acrylamides, such as N-cyclohexylacrylamide and N-tert-butylacrylamide, are rare examples of polymers showing composition-dependent thermoresponsive behavior in water. They show unexpected behavior different from the conventional copolymers of a thermoresponsive polymer with hydrophobic comonomers. On increasing the amount of the hydrophobic comonomer in the polymer, there is a change from UCST-type phase behavior to complete solubility, followed by compositions showing LCST-type transition and insolubility in water (U-S-L-I-type change). At 50 mol% of less hydrophobic N-tert-butyl acrylamide (logP 0.87) in contrast to ≈25 mol% of N-cyclohexylacrylamide (logP 1.51), the broad UCST-type transition of polymethacrylamide is turned to LCST-type transitions with very narrow hysteresis in water and PBS buffer. The use of H-bond and water-structure breaker showed the role of H-bonding and hydrophobic interactions in phase transitions of UCST-type and LCST-type, respectively. This work is an important add-on to the infant field of nonionic polymers of UCST-type.

Low-Density Self-Assembled Poly(N-Isopropyl Acrylamide) Sponges with Ultrahigh and Extremely Fast Water Uptake and Release.

Poly(N-isopropyl acrylamide) (PNIPAM) hydrogels are well known for their temperature-dependent water uptake and release. Hence, they are ideal candidates for water management applications. However, efficiency and rate of water uptake and release, respectively, have to be optimized. Here, highly stable 3D PNIPAM sponges that show a sufficiently low density and high specific pore volume, required for maximizing the amount and rate of water absorption-desorption, are presented. They are prepared by a top-down approach based on freeze-drying a dispersion of short crosslinked PNIPAM fibers coated with crosslinked PNIPAM. The sponges have low densities (4.10-21.04 mg cm-3 ), high porosities >98%, and high specific pore volumes in the range of 47-243 cm3 g-1 depending on the concentration of the dispersions. The sponges absorb high amounts of water (≈7000%) at temperatures below the lower critical solution temperature (LCST) of PNIPAM and can release more than 80% of the absorbed water above the LCST in less than 2 min. Moreover, the water-swollen sponges are reversibly foldable, can be confined to different shapes, and have compressive elastic modulus below 10 Pa. Hence, these spongy materials are of interest not only for water management but also for biomedical applications, smart textiles, and catalysis.

Composite Polymeric Membranes with Directionally Embedded Fibers for Controlled Dual Actuation.

In this paper, preparation method and actuation properties of an innovative composite membrane composed of thermo- and pH-responsive poly(N-isopropylacrylamide-co-acrylic acid) fibers (average diameter ≈ 905 nm) embedded within a passive thermoplastic polyurethane (TPU) matrix at different angles with degree of alignment as high as 98% are presented. The composite membrane has a gradient of TPU along the thickness. It has the capability of temperature- and pH-dependent direction-, and size-controlled actuation in few minutes. The stresses generated at the responsive fiber and nonresponsive matrix provide actuation, whereas the angle at which fibers are embedded in the matrix controls the actuation direction and size. The temperature has no effect on actuation and actuated forms at pH 7 and above, whereas the size of the actuated forms can be controlled by the temperature at lower pH. The membranes are strong enough to reversibly lift and release ≈426 times weight of their own mass (2.47 g metal ring is lifted by a 5.8 mg membrane). Soft actuators are of interest as smart scaffolds, robotics, catalysis, drug release, energy storage, electrodes, and metamaterials.

Exploration of Macroporous Polymeric Sponges As Drug Carriers.

Achieving high drug loading capacity and controlling drug delivery are two main challenges related to drug carriers. In this study, polymeric macroporous sponges with very high pore volume and large porosity are introduced as a new-type of drug carrier. Due to the high pore volume (285 and 166 cm3/g for the sponges with densities of 3.5 and 6.0 mg/cm3, respectively), the sponges exhibit very high drug loading capacities with average values of 1870 ± 114 and 2697 ± 73 mg/g in the present study, which is much higher than the meso and microporous drug carriers (<1500 mg/g). In order to control the release profiles, an additional poly(p-xylylene) (PPX) coating was deposited by chemical vapor deposition on the drug loaded sponge. Consequently, Artemisone (ART) release in the aqueous medium could be retarded, depending on the density of the sponge and the thickness of the coating. In future, the new 3D polymeric sponges would be highly beneficial as drug carriers for the programmed release of drugs for treatment of chronic diseases.

Compaction and Transmembrane Delivery of pDNA: Differences between l-PEI and Two Types of Amphiphilic Block Copolymers.

Polycations are popular agents for nonviral delivery of DNA to mammalian cells. Adding hydrophobic, biodegradable, or cell-penetrating functions could help to improve their performance, which at present is below that of viral agents. A crucial first step in gene delivery is the complexation of the DNA. The characteristics of these "polyplexes" presumably influence or even determine the subsequent steps of membrane passage, intracellular traveling/DNA release, and nuclear uptake. Herein, polyplexes formed with linear poly(ethylenimine) (l-PEI) are compared to complexes generated with functionalized diblock copolymers. While l-PEI interacts only electrostatically with the DNA, interaction in the case of the diblock polymers may be mixed-mode. In certain cases, transfection efficiency improved when the polyplexes were formed in hypertonic solution. Moreover, whereas conventional PEI-based polyplexes enter the cells via endocytosis, at least one of the diblock agents seemed to promote entry via transient destabilization of the plasma membrane.

Self-Rolled Porous Hollow Tubes Made up of Biodegradable Polymers.

A tubular highly porous scaffold of polylactide (PLA) and poly-ε-caprolactone (PCL) is fabricated by self-rolling of a 2D fibrous bilayer of PLA and PCL in water without use of any classical thermo-/pH-responsive polymers. The self-rolling and diameter of the tube are dependent upon the bilayer thickness and temperature. A 75 µm thick 2D bilayer (PLA = 25 µm; PCL = 50 µm) rolls to a hollow tube of diameter around 0.41 mm with multilayered wall at 40 °C within 5 min. The tubes keep their form and size in water at all temperatures once they are formed. The interesting properties of the hollow tubes, that is, permeation of gases through the walls and flow of water without leakage under tested conditions in combination with good mechanical stability, use of only biodegradable polymers, and easy and reproducible fabrication method, allow them to be promising candidates for future studies as scaffolds for tissue engineering.

Poly(amino acid)-Based Gel Fibers with pH Responsivity by Coaxial Reactive Electrospinning.

Electrospinning is a well-known technique for the preparation of scaffolds for biomedical applications. In this work, a continuous electrospinning method for gel fiber preparation is presented without a spinning window. As proof of concept, the preparation of poly(aspartic acid)-based hydrogel fibers and their properties are described by using poly(succinimide) as shell polymer and 2,2,4(2,4,4)-trimethyl-1,6-hexanediamine as cross-linker in the core of the nozzle. Cross-linking takes place as the two solutions get in contact at the tip of the nozzle. The impact of solution concentrations and feeding rates on fiber morphology, proof of the presence of cross-links as well as pH sensitivity after the transformation of the poly(succinimide)-based material to poly(aspartic acid) is presented.

Self-Organization of Gold Nanoparticle Assemblies with 3D Spatial Order and Their External Stimuli Responsiveness.

Gold nanoparticles (AuNP) with pyridyl end-capped polystyrenes (PS-4VP) as "quasi-monodentate" ligands self-assemble into ordered PS-4VP/AuNP nanostructures with 3D hexagonal spatial order in the dried solid state. The key for the formation of these ordered structures is the modulation of the ratio AuNP versus ligands, which proves the importance of ligand design and quantity for the preparation of novel ordered polymer/metal nanoparticle conjugates. Although the assemblies of PS-4VP/AuNP in dispersion lack in high dimensional order, strong plasmonic interactions are observed due to close contact of AuNP. Applying temperature as an external stimulus allows the reversible distortion of plasmonic interactions within the AuNP nanocomposite structures, which can be observed directly by naked eye. The modulation of the macroscopic optical properties accompanied by this structural distortion of plasmonic interaction opens up very interesting sensoric applications.

LCST and UCST in One: Double Thermoresponsive Behavior of Block Copolymers of Poly(ethylene glycol) and Poly(acrylamide-co-acrylonitrile).

The change in thermoresponsive behavior from a single phase transition of upper critical solution temperature (UCST)-type of an acrylamide-acrylonitrile copolymer (AAm-co-AN) to a double responsive behavior (LCST-UCST-type (LCST, lower critical solution temperature)) in water by the introduction of a poly(ethylene glycol) (PEG) block is highlighted in the present work. The polymer is synthesized in a simple way by free-radical polymerization of acrylamide and acrylonitrile using a poly(ethylene glycol) (PEG) macro-azoinitiator. The dual thermoresponsive behavior was observed in a wide range of concentrations repeatable for many cycles with very small hysteresis depending upon the ratio of AAm, AN and PEG. Static light scattering (SLS) and dynamic light scattering (DLS) together with turbidity photometry and transmission electron microscopy confirmed a unique phase transition behavior due to the temperature dependent change in the morphology from micelles to agglomerates. The low cytotoxicity and two-in-one thermoresponsive behavior makes the polymer promising for biomedical applications in the future.

A rare example of the formation of polystyrene-grafted aliphatic polyester in one-pot by radical polymerization.

The radical copolymerization of cyclic ester ß-propiolactone (ß-PL) with styrene (St) at 120 °C, with a complete range of monomer ratios, is a rare example of a system providing graft copolymers (PSt-g-ß-PL) in one pot. The structure of the resulting ß-PL-St copolymers was proven by using a combination of different characterization techniques, such as 1D and 2D NMR spectroscopy and gel permeation chromatography (GPC), before and after alkaline hydrolysis of the polymers. The number of grafting points increased with an increasing amount of ß-PL in the feed. A significant difference in the reactivity of St and ß-PL and radical chain-transfer reactions at the polystyrene (PSt) backbone, followed by combination with the active growing poly(ß-PL) chains, led to the formation of graft copolymers by a grafting-onto mechanism.

Tea-bag-like polymer nanoreactors filled with gold nanoparticles.

Gold-containing polymer nanotubes, which showed both catalytic activity and resistance to leaching, were prepared by the "tubes by fiber templates" (TUFT) process. For this purpose, electrospun polymer nonwovens with incorporated poly(L-lactide)-stabilized gold nanoparticles were coated with poly(p-xylylene) by the chemical vapor deposition process, and then the inner fiber templates were removed. The resulting polymer tubes carried encapsulated gold nanoparticles which were shown to be immobilized and featured pronounced catalytic activity towards the hydrolytic oxidation of dimethylphenylsilane and the alcoholysis of dimethylphenylsilane with n-butanol. The macroscopic nonwovens could be used as tea-bag-like catalyst systems and showed excellent reusability.

Chronic poly(l-lactide) (PLA)- microplastic ingestion affects social behavior of juvenile European perch (Perca fluviatilis).

Juvenile perch were exposed to 2 % (w/w) poly(l-lactide) (PLA) microplastic particles (90-150 µm) in food pellets, or 2 % (w/w) kaolin particles, and a non-particle control food over 6 months. Chronic ingestion of PLA microplastics significantly affected the social behavior of juvenile perch, evident as a significantly increased reaction to the vision of conspecifics. PLA ingestion did not alter life cycle parameters, or gene expression levels. In addition to reactions to conspecifics, fish that ingested microplastic particles showed tendencies to decrease locomotion, internal schooling distance, and active predator responses. The ingestion of natural particles (kaolin) significantly downregulated the expression of genes related to oxidative stress and androgenesis in the liver of juvenile perch, and we found tendencies to downregulated expression of genes related to xenobiotic response, inflammatory response, and thyroid disruption. The present study demonstrated the importance of natural particle inclusion and the potential behavioral toxicity of one of the commercially available biobased and biodegradable polymers.