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.

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.

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.

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.

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.

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.

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.

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.

Matrix supported poly(2-oxazoline)-based hydrogels for DNA catch and release.

We describe the synthesis of matrix supported hydrogel structures based on amine containing poly(2-oxazoline)s and their use to bind and release genetic material for potential applications in diagnostics or pathogen detection. Amine containing poly(2-oxazoline)s were synthesized by copolymerization of 2-ethyl-2-oxazoline with a monomer bearing a tert-butyl oxycarbonyl (Boc) protected amine group in the 2-position and subsequent deprotection. The statistical copolymers were used to generate hydrogels and matrix supported hydrogels by cross-linking of a certain fraction of the amine groups with epichlorhydrin. Supported structures were prepared by soaking porous polyethylene (PE) or polypropylene (PP) filter materials in a copolymer/epichlorhydrin solution, which was cross-linked upon heating. Scanning electron microscopy (SEM) of the composites revealed a bead like structure of the gel phase, which could be attributed to a lower critical solution temperature (LCST) behavior of the initial polymer prior to gelation. The dependency of the LCST behavior on the content of amine groups was investigated. Swelling values and the ratio of hydrogel per composite was determined using water sorption analysis. Subsequently, the ability of the systems to absorb and release labeled DNA was tested. Uptake and stimulated release, triggered by changes in pH, temperature, and heparin concentration, were investigated using fluorescence microscopy. Polymerase chain reaction (PCR) proved the successful recovery of the DNA, demonstrating the potential of the presented system for a broad range of molecular biological applications.

Linear poly(ethylene imine)-based hydrogels for effective binding and release of DNA.

A series of copolymers containing both amine groups of linear poly(ethylene imine) (LPEI) and double bonds of poly(2-(3-butenyl)-2-oxazoline) (PButEnOx) was prepared. To this end, a poly(2-ethyl-2-oxazoline) (PEtOx) precursor was hydrolyzed to the respective LPEI and functionalized in an amidation reaction with butenyl groups resulting in the double bond containing poly(2-(3-butenyl-2-oxazoline)-co-ethylene imine) (P(ButEnOx-co-EI)). Hydrogels were obtained by cross-linking with dithiols under UV-irradiation resulting in networks with different properties in dependence of the content of double bonds. The developed method allows the exact control of the amount of ethylene imine units within the copolymer and, thus, within the resulting hydrogels. The gel structures were characterized by solid state NMR and infrared spectroscopy. In addition the water uptake behavior from the liquid and the gas phase was investigated. It was shown by an ethidium bromide assay (EBA) that the copolymers and the respective hydrogels were able to bind and release DNA. Furthermore, the influence of the ethylene imine content on this interaction was investigated.

Small but powerful: co-assembly of polyether-based triblock terpolymers into sub-30 nm micelles and synergistic effects on cellular interactions.

We introduce a versatile ABC triblock terpoly- mer platform based on poly(ethylene oxide)-block-poly(allyl glycidyl ether)-block-poly(tert-butyl glycidyl ether) (PEO-b-PAGE-b-PtBGE) and subsequent functionalization of the PAGE segment with thiogalactose (hydroxyl), cysteamine (amino), and 2-mercaptopropionic acid (carboxy) by thiol-ene chemistry. These materials are used to prepare core-shell-corona micelles with a PtBGE core, a PAGE shell, and a PEO corona and sizes below 30 nm in aqueous media. We investigate the influence of different functional groups on micelle formation and cellular uptake. Moreover, co-assembly of differently functionalized materials allows to create micelles with a mixed shell and adjustable charge and, in that way, important characteristics such as cell uptake or cytotoxicity can be controlled. Furthermore, we demonstrate that even the uptake mechanism depends on the substitution pattern of the underlying triblock terpolymer.

Poly(2-oxazoline) functionalized surfaces: from modification to application.

Poly(2-oxazoline)s (POxs) are a versatile class of biocompatible polymers, which have been investigated as poly(ethylene glycol) (PEG) alternatives. In recent years, POxs have drawn significant attention as coatings for antifouling applications. In this tutorial review different approaches to immobilize POxs on surfaces as well as properties and applications of POx coated surfaces will be presented.

Surface functionalized cryogels - characterization methods, recent progress in preparation and application.

Cryogels are polymeric materials with a sponge-like microstructure and have attracted significant attention in recent decades. Research has focused on their composition, fabrication techniques, characterization methods as well as potential or existing fields of applications. The use of functional precursors or functionalizing ligands enables the preparation of cryogels with desired properties such as biocompatibility or responsivity. They can also exhibit adsorptive properties or can be used for catalytical purposes. Although a very brief overview about several functional (macro-)monomers and functionalizing ligands has been provided by previous reviewers for certain cryogel applications, so far there has been no particular focus on the evaluation of the functionalization success and the characterization methods used. This review will provide a comprehensive overview of different characterization methods most recently used for the evaluation of cryogel functionalization. Furthermore, new functional (macro-)monomers and subsequent cryogel functionalization strategies are discussed, based on synthetic polymers, biopolymers and a combination of both. This review highlights the importance of the functionalization aspect in cryogel research in order to produce materials with tailored properties for certain applications.

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.

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).

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).

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.