Cryogels Based on Poly(2-oxazoline)s through Development of Bi- and Trifunctional Cross-Linkers Incorporating End Groups with Adjustable Stability.

1,4-Bis(iodomethyl)benzene and 1,3,5-tris(iodomethyl)benzene were used as initiators for the cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline (EtOx) and its copolymerization with tert-butyl (3-(4,5-dihydrooxazol-2-yl)propyl)carbamate (BocOx) or methyl 3-(4,5-dihydrooxazol-2-yl)propanoate (MestOx). Kinetic studies confirmed the applicability of these initiators. Termination with suitable nucleophiles resulted in two- and three-armed cross-linkers featuring acrylate, methacrylate, piperazine-acrylamide, and piperazine-methacrylamide as polymerizable ω-end groups. Matrix-assisted laser desorption/ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed the successful attachment of the respective ω-end groups at all initiation sites for every prepared cross-linkers. Except for acrylate, each ω-end group remained stable during deprotection of BocOx containing cross-linkers. The cryogels were prepared using EtOx-based cross-linkers, as confirmed by solid-state NMR spectroscopy, scanning electron microscopy, and thermogravimetric analysis. Stability tests revealed a complete dissolution of the acrylate-containing gels at pH = 14, whereas the piperazine-acrylamide-based cryogels featured excellent hydrolytic stability.

Determination of ω-end functionalities in tailored poly(2-alkyl-2-oxazoline)s by liquid chromatography and mass spectrometry.

The in-depth analytical characterization of polymers, in particular regarding intended biomedical applications, is becoming increasingly important to elucidate their structure-property relationships. Specifically, end group analysis of e.g. polymers featuring a 'stealth effect' towards the immune system is of particular importance because of their use in coupling reactions to bioactive compounds. Herein, we established a liquid chromatography (LC) protocol to analyse bicyclo[6.1.0]nonyne-functionalized poly(2-alkyl-2-oxazoline)s (POx)s as promising functional polymers that can be applied in strain-promoted click reactions. This work involved the synthesis of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) by living cationic ring-opening polymerization (CROP) with different molar masses ranging from 2 up to 17.5 kDa and, to our knowledge, the first liquid chromatographic analysis of PMeOx. The developed analytical protocol enables the quantitative determination of post-polymerization reaction sequences with respect to the conversion of the ω-end groups. All synthesized polymers were straightforwardly analysed on a C18-derivatized silica monolithic column under reversed-phase chromatographic conditions with a binary mobile phase gradient comprising a mixture of acetonitrile and water. Subsequent mass spectrometry of collected elution fractions enabled the confirmation of the desired ω-end group functionalities and the identification of synthetic by-products.

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d-fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru-bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d-fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).

Evaluation of reproducible cryogel preparation based on automated image analysis using deep learning.

Cryogels represent a class of porous sponge-like materials possessing unique properties including high-fidelity reproduction of tissue structure and maximized permeability. Their architecture is mainly based on an interconnected network of macropores that provides sufficient stability while allowing the movement of substances through the material. In most cryogel applications, the pore size is very important, especially when the material is used as a 3D scaffold for tissue culture, applied as a filter, or utilized as a membrane. In this study, poly(dimethylacrylamide-co-2-hydroxyethyl methacrylate) cryogels have been prepared by two preparation methods to investigate the reproducibility of homogeneous pore structures and pore sizes. Automated image analysis algorithms were developed to rapidly evaluate cryogel pore sizes based on scanning electron microscopy (SEM) images. The quantification approach contained a unique combination of classical and deep learning-based algorithms. To validate the accuracy of the two models, we compared the results obtained from automated SEM image analysis with those from manual pore size determinations and mercury intrusion porosimetry (MIP) measurements. Effect sizes were calculated to compare the results from manual and automated pore size measurements for the cryogel reproducibility series. 81% of the values obtained revealed only trivial differences, which strongly suggests that automated image analysis can reliably substitute the manual evaluation of cryogel pore sizes. The use of an adapted reactor setup yielded cryogels with heterogeneous morphologies in the absence of recognizable pore structures. With the conventional cryogel preparation using plastic syringes, the obtained cryogels represented highly reproducible morphologies and pore sizes in the range between 17 and 22 µm. Calculated effect sizes within the cryogel replicate series revealed only trivial differences between the obtained pore sizes in 83.5% or 99.4% of the data (classical approach and deep learning-based approach, respectively).

Dimethylsulfoniopropionate decorated cryogels as synthetic spatially structured habitats of marine bacterial communities.

In microbial consortia bacteria often settle on other organisms that provide nutrients and organic material for their growth. This is true for the plankton where microalgae perform photosynthesis and exude metabolites that feed associated bacteria. The investigation of such processes is difficult since algae provide bacteria with a spatially structured environment with a gradient of released organic material that is hard to mimic. Here we introduce the design and synthesis of a cryogel-based microstructured habitat for bacteria that provides dimethylsulfoniopropionate (DMSP) as a carbon and sulfur source for growth. DMSP, a widely distributed metabolite released by algae, is thereby made available for bacteria in a biomimetic manner. Based on a novel DMSP derived building block (DMSP-HEMA), we synthesized cryogels providing structured surfaces for settlement and delivering the organic material fueling bacterial growth. By monitoring bacterial settlement and performance we show that the cryogels represent microbial arenas mimicking the ecological situation in the plankton.

From Light to Structure: Photo Initiators for Radical Two-Photon Polymerization.

Two-photon polymerization (2PP) represents a powerful technique for the fabrication of precise three-dimensional structures on a micro- and nanometer scale for various applications. While many review articles are focusing on the used polymeric materials and their application in 2PP, in this review the class of two-photon photo initiators (2PI) used for radical polymerization is discussed in detail. Because the demand for highly efficient 2PI has increased in the last decades, different approaches in designing new efficient 2PIs occurred. This review summarizes the 2PIs known in literature and discusses their absorption behavior under one- and two-photon absorption (2PA) conditions, their two-photon cross sections (σTPA ) as well as their efficiency under 2PP conditions. Here, the photo initiators are grouped depending on their chromophore system (D-π-A-π-D, D-π-D, etc.). Their polymerization efficiencies are evaluated by fabrication windows (FW) depending on different laser intensities and writing speeds.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Poly(2-ethyl-2-oxazoline) Featuring a Central Amino Moiety.

The incorporation of an amino group into a bifunctional initiator for the cationic ring-opening polymerization (CROP) is achieved in a two-step reaction. Detailed kinetic studies using 2-ethyl-2-oxazoline demonstrate the initiators' eligibility for the CROP yielding well-defined polymers featuring molar masses of about 2000 g mol-1 . Deprotection of the phthalimide moiety subsequent to polymerization enables the introduction of a cyclooctyne group in central position of the polymer which is further exploited in a strain-promoted alkyne-azide click reaction (SpAAC) with a Fmoc-protected azido lysine representing a commonly used binding motif for site specific polymer-protein/peptide conjugation. In-depth characterization via electrospray ionization mass spectrometry (ESI) confirms the success of all post polymerization modification steps.

Core-Cross-linked Fluorescent Worm-Like Micelles for Glucose-Mediated Drug Delivery.

We herein report the fabrication of core-crosslinked, fluorescent, and surface-functionalized worm-like block copolymer micelles as drug delivery vehicles. The polyether-based diblock terpolymer [allyl-poly(ethylene oxide)-block-poly(2-ethylhexyl glycidyl ether-co-furfuryl glycidyl ether)] was synthesized via anionic ring opening polymerization, and self-assembly in water as a selective solvent led to the formation of long filomicelles. Subsequent cross-linking was realized using hydrophobic bismaleimides as well as a designed fluorescent cross-linker for thermally induced Diels-Alder reactions with the furfuryl units incorporated in the hydrophobic block of the diblock terpolymer. As a fluorescent cross-linker, we synthesized and incorporated a cyanine 5-based bismaleimide in the cross-linking process, which can be used for fluorescence tracking of the particles. Furthermore, we covalently attached glucose to the allyl end groups present on the surface of the micelles to investigate active glucose-mediated transport into suitable cell lines. First studies in 2D as well as 3D cell culture models suggest a glucose-dependent uptake of the particles into cells despite their unusually large size compared to other nanoparticle systems used in drug delivery.

Two-Photon Polymerized Poly(2-Ethyl-2-Oxazoline) Hydrogel 3D Microstructures with Tunable Mechanical Properties for Tissue Engineering.

The main task of tissue engineering (TE) is to reproduce, replicate, and mimic all kinds of tissues in the human body. Nowadays, it has been proven useful in TE to mimic the natural extracellular matrix (ECM) by an artificial ECM (scaffold) based on synthetic or natural biomaterials to regenerate the physiological tissue/organ architecture and function. Hydrogels have gained interest in the TE community because of their ability to absorb water similar to physiological tissues, thus mechanically simulating the ECM. In this work, we present a novel hydrogel platform based on poly(2-ethyl-2-oxazoline)s, which can be processed to 3D microstructures via two-photon polymerization (2PP) with tunable mechanical properties using monomers and crosslinker with different degrees of polymerization (DP) for future applications in TE. The ideal parameters (laser power and writing speed) for optimal polymerization via 2PP were obtained using a specially developed evaluation method in which the obtained structures were binarized and compared to the computer-aided design (CAD) model. This evaluation was performed for each composition. We found that it was possible to tune the mechanical properties not only by application of different laser parameters but also by mixing poly(2-ethyl-2-oxazoline)s with different chain lengths and variation of the crosslink density. In addition, the swelling behavior of different fabricated hydrogels were investigated. To gain more insight into the viscoelastic behavior of different fabricated materials, stress relaxation tests via nanoindentation experiments were performed. These new hydrogels can be processed to 3D microstructures with high structural integrity using optimal laser parameter settings, opening a wide range of application properties in TE for this material platform.

Improved Bioactivity of the Natural Product 5-Lipoxygenase Inhibitor Hyperforin by Encapsulation into Polymeric Nanoparticles.

Hyperforin, a highly hydrophobic prenylated acylphloroglucinol from the medical plant St. John's Wort, possesses anti-inflammatory properties and suppresses the formation of proinflammatory leukotrienes by inhibiting the key enzyme 5-lipoxygenase (5-LO). Despite its strong effectiveness and the unique molecular mode of interference with 5-LO, the high lipophilicity of hyperforin hampers its efficacy in vivo and, thus, impairs its therapeutic value, especially because of poor water solubility and strong plasma (albumin) protein binding. To overcome these hurdles that actually apply to many other hydrophobic 5-LO inhibitors, we have encapsulated hyperforin into nanoparticles (NPs) consisting of acetalated dextran (AcDex) to avoid plasma protein binding and thus improve its cellular supply under physiologically relevant conditions. Encapsulated hyperforin potently suppressed 5-LO activity in human neutrophils, but it failed to interfere with 5-LO activity in a cell-free assay, as expected. In the presence of human serum albumin (HSA), hyperforin was unable to inhibit cellular 5-LO activity, seemingly because of strong albumin binding. However, when encapsulated into NPs, hyperforin caused strong inhibition of 5-LO activity in the presence of HSA. Together, encapsulation of the highly hydrophobic hyperforin as a representative of lipophilic 5-LO inhibitors into AcDex-based NPs allows for efficient inhibition of 5-LO activity in neutrophils in the presence of albumin because of effective uptake and circumvention of plasma protein binding.

Artificial Microbial Arenas: Materials for Observing and Manipulating Microbial Consortia.

From the smallest ecological niche to global scale, communities of microbial life present a major factor in system regulation and stability. As long as laboratory studies remain restricted to single or few species assemblies, however, very little is known about the interaction patterns and exogenous factors controlling the dynamics of natural microbial communities. In combination with microfluidic technologies, progress in the manufacture of functional and stimuli-responsive materials makes artificial microbial arenas accessible. As habitats for natural or multispecies synthetic consortia, they are expected to not only enable detailed investigations, but also the training and the directed evolution of microbial communities in states of balance and disturbance, or under the effects of modulated stimuli and spontaneous response triggers. Here, a perspective on how materials research will play an essential role in generating answers to the most pertinent questions of microbial engineering is presented, and the concept of adaptive microbial arenas and possibilities for their construction from particulate microniches to 3D habitats is introduced. Materials as active and tunable components at the interface of living and nonliving matter offer exciting opportunities in this field. Beyond forming the physical horizon for microbial cultivates, they will enable dedicated intervention, training, and observation of microbial consortia.

TBD-Catalyzed Ring-Opening Polymerization of Alkyl-Substituted Morpholine-2,5-Dione Derivatives.

In a two-step synthesis, five different alkyl-substituted morpholine-2,5-dione monomers were synthesized from the natural amino acids glycine, alanine, valine, leucine, and isoleucine. The heterocyclic compounds crystallize in a boat-like conformation and are polymerized via 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-catalyzed ring-opening polymerization (ROP) in tetrahydrofuran. Well-defined polymers could be obtained from the monomers based on valine, leucine, and isoleucine at a feed ratio of M/I/TBD = 100/1/0.5. Kinetic studies of the ROP reveal that the molar masses and dispersities (D < 1.2) could be well controlled, as confirmed by size exclusion chromatography and 1 H NMR spectroscopy. At conversions above 50%, the polymerization rate decreases and the dispersity slightly increases, presumably due to transesterification. Matrix-assisted laser desorption time-of-flight mass spectrometry indicates the presence of polymer chains with α-end groups derived from the initiator.

Spherical and Worm-Like Micelles from Fructose-Functionalized Polyether Block Copolymers.

This paper presents the synthesis and characterization of d-fructose modified poly(ethylene glycol) (Fru-PEG) and fructose modified poly(ethylene glycol)-block-poly(ethyl hexyl glycidyl ether) (Fru-PEG-b-PEHG) that are both prepared by initiation with isopropyliden protected fructose, followed by deprotection of the sugar. The block copolymers are self-assembled into micelles, and are subsequently characterized by cryo-TEM and dynamic light scattering. The fluorescent dye Nile red is encapsulated as a model hydrophobic compound and fluorescent marker to perform initial uptake tests with breast cancer cells. The uptake of sugar and nonsugar decorated micelles is compared.

Photocontrolled Release of Chemicals from Nano- and Microparticle Containers.

A benzoin-derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well-controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano- and microparticle containers was performed by confocal laser scanning microscopy in a proof-of-principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV-sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self-healing processes.

Curcuminoid-BF2 complexes: Synthesis, fluorescence and optimization of BF2 group cleavage.

Eight difluoroboron complexes of curcumin derivatives carrying alkyne groups containing substituents have been synthesized following an optimised reaction pathway. The complexes were received in yields up to 98% and high purities. Their properties as fluorescent dyes have been investigated. Furthermore, a strategy for the hydrolysis of the BF2 group has been established using aqueous methanol and sodium hydroxide or triethylamine.

Synthesis of d-fructose conjugated ligands via C6 and C1 and their corresponding [Ru(bpy)2(L)]Cl2 complexes.

A pyridyl triazole (pyta) modified sucrose ligand was prepared in a seven step synthesis using d-glucose as the protection group for d-fructose and starting from commercially available sucrose. After complexation with Ru(bpy)2Cl2 precursor, the sucrose-conjugated Ru complex of the general formula [Ru(bpy)2(L)]Cl2 was formed. Acidic cleavage of the d-glucose unit led to the first d-fructose conjugated metal complex viad-fructose C6 in literature. Additionally, pyta-modified d-fructose via C1 and the corresponding Ru complex were synthesized. All compounds were analyzed by Rf values, specific rotation, NMR, IR, UV/Vis and fluorescence spectroscopy, mass spectrometry and elemental analysis.

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting.

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent.

Radiolabeling of DOTA-like conjugated peptides with generator-produced (68)Ga and using NaCl-based cationic elution method.

Gallium-68 ((68)Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize (68)Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system,(68)Ga(3+) of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3-4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of (68)Ga radiopharmaceuticals (12-16 min).

Antibacterial effect of silver (I) carbohydrate complexes on oral pathogenic key species in vitro.

BACKGROUND: It was the aim of this study to evaluate the antibacterial impact of two silver(I) carbohydrate complexes with tripodal thioglycosides, namely tris[2-(ß-D-thio-glucopyranosyl)ethyl]-amine-silver(I)-nitrate (3) and tris[2-(α-D-thio-manno-pyranosyl)ethyl]-amine-silver(I)-nitrate (4), on five oral pathogenic bacterial strains. Furthermore, cytocompatibility was tested using human gingival fibroblasts (HGF). METHODS: Minimum inhibitory concentrations (MIC) were determined on five oral pathogenic bacterial strains by using the broth microdilution method: Fusobacterium nucleatum (ATCC 10953), Aggregatibacter actinomycetemcomitans (ATCC 33384), Porphyromonas gingivalis (ATCC 33277), Streptococcus mutans (ATCC 25175) and Enterococcus faecalis (DSMZ 20376). Furthermore, antimicrobial efficiency was tested using agar diffusion assays. To evaluate cytocompatibility, human gingival fibroblasts (HGFs) were exposed to AgNO3 and complex 3 followed by a live/dead staining. RESULTS: MIC of the silver(I) complexes ranged between 0.625 and 5.0 mmol/L. The silver complexes 3 and 4 showed higher antibacterial efficiency against all tested species than AgNO3. Antibacterial efficiency of complexes 3 and 4 on F. nucleatum (≥18 mm) and A. actinomycetemcomitans (≥23 mm) was more pronounced than against P. gingivalis (≥15 mm). Complex 3 (20 mM) induced the largest inhibition zones (30 to 31 mm) on Gram-negative strains. For Gram-positive strains, the largest inhibition zones were achieved by complex 3 (20 mM/S. mutans: 28 mm, E. faecalis: 18 mm). Complex 3 had a lower cytotoxic impact on HGFs compared to AgNO3 by the power of ten. CONCLUSIONS: The findings suggest that silver(I) carbohydrate complexes 3 and 4 might function as novel antimicrobial agents for the treatment of periodontal, carious or endodontic diseases.