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    Boosting the Cell Harvesting Performance of Poly(di(ethylene glycol)methyl ether methacrylate) Cell Release Layers via Copolymerization of Photo‐ and Thermoresponsive Monomers
    The performance of the cell‐selective thermoresponsive poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) cell harvest system is shown to be drastically enhanced by exploiting the combination of photoresponsive spiropyran derivates and PDEGMA in copolymerized brushes. The analysis of copolymerized 1′‐(2‐methacryloxyethyl)‐3′,3′‐dimethyl‐6‐nitrospiro(2 H ‐1‐benzopyran‐2,2′‐indoline) (SPMA) (DEMGA) di(ethylene glycol)methyl ether methacrylate brushes revealed that a minor adjustment of the SPMA/DEGMA ratios results in a significant alternation of wettability as well as protein adsorption, when switching the temperature from 37 to 22 °C and alternately irradiating using different light wavelengths (from 530 to 365 nm). Thin P(SPMA‐ co ‐DEGMA) layers supported pancreatic tumor PaTu 8988t cells with high cell viability. Copolymer layers with 2.5% SPMA/DEGMA led to the highest efficiency of enzyme‐free cell release with very good cell viability. The release is induced by cooling the cell culture medium to 22 °C and irradiating the surface with 365 nm light. Compared to neat PDEGMA, the P(SPMA‐ co ‐DEGMA) layers showed a threefold increase in the speed of the change of cell morphology of the attached cells and a >5 times increased fraction of detached cells, which underlines the potential of these dual responsive PDEGMA systems for optimized performance in the facile capture, culture, and release of different cell lines.
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    Fluorimetric Cell Analysis with 9‐Aryl‐Substituted Berberine Derivatives as DNA‐Targeting Fluorescent Probes
    DNA‐sensitive fluorescent light‐up probes based on berberine are presented. This biogenic fluorophore was chosen as central unit to use its potential biocompatibility and its DNA‐binding properties. To provide predictable fluorescence quenching in aqueous solution and a fluorescence light‐up effect upon DNA binding, aryl substituents were attached at the 9‐position by Suzuki‐Miyaura coupling reactions. The 9‐arylberberine derivatives have a very low fluorescence quantum yield ( Φ fl =<0.02), which is caused by the radiationless deactivation of the excited state by torsional relaxation about the biaryl axis. In addition, these berberine derivatives intercalate into DNA with high affinity ( K b =2.0−22×10 4 M −1 ). Except for the nitrophenyl‐ and hydroxyphenyl‐substituted derivatives, all tested compounds exhibited a pronounced fluorescence light‐up effect upon association with DNA, because the deactivation of the excited‐state by torsional relaxation is suppressed in the DNA binding site. Most notably, it was shown exemplarily with the 9‐(4‐methoxyphenyl)‐ and the 9‐(6‐methoxynaphthyl)‐substituted derivatives that these properties are suited for fluorimetric cell analysis. In particular, these probes generated distinct staining patterns in eukaryotic cells (NIH 3T3 mouse fibroblasts), which enabled the identification of nuclear substructures, most likely heterochromatin or nucleoli, respectively.
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    Mild Quantitative One Step Removal of Macrophages from Cocultures with Human Umbilical Vein Endothelial Cells Using Thermoresponsive Poly(Di(Ethylene Glycol)Methyl Ether Methacrylate) Brushes
    The authors report on a mild, label‐free, and fast method for the separation of human umbilical vein endothelial cells (HUVEC), which are relevant cells, whose use is not limited to studies of endothelial dysfunction, from cocultures with macrophages to afford HUVEC in ≈100% purity. Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes with a dry thickness of (5 ± 1) nm afford the highly effective one‐step separation by selective HUVEC detachment, which is based on the brushes' thermoresponsive behavior. Below the thermal transition at 32 °C the brushes swells and desorbs attached proteins, resulting in markedly decreased cell adhesion. Specifically, HUVEC and macrophages, which are differentiated from THP‐1 monocytes, are seeded and attached to PDEGMA brushes at 37°C. After decreasing the temperature to 22°C, HUVEC shows a decrease in their cell area, while the macrophages are not markedly affected by the temperature change. After mild flushing with a cell culture medium, the HUVEC can be released from the surface and reseeded again with ≈100% purity on a new surface. With this selective cell separation and removal method, it is possible to separate and thereby purify HUVEC from macrophages without the use of any releasing reagent or expensive labels, such as antibodies.
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    On‐Demand Cell Sheet Release with Low Density Peptide‐Functionalized Non‐LCST Polymer Brushes
    Cell sheet harvesting offers a great potential for the development of new therapies for regenerative medicine. For cells to adhere onto surfaces, proliferate, and to be released on demand, thermoresponsive polymeric coatings are generally considered to be required. Herein, an alternative approach for the cell sheet harvesting and rapid release on demand is reported, circumventing the use of thermoresponsive materials. This approach is based on the end‐group biofunctionalization of non‐thermoresponsive and antifouling poly(2‐hydroxyethyl methacrylate) (p(HEMA)) brushes with cell‐adhesive peptide motifs. While the nonfunctionalized p(HEMA) surfaces are cell‐repellant, ligation of cell‐signaling ligand enables extensive attachment and proliferation of NIH 3T3 fibroblasts until the formation of a confluent cell layer. Remarkably, the formed cell sheets can be released from the surfaces by gentle rinsing with cell‐culture medium. The release of the cells is found to be facilitated by low surface density of cell‐adhesive peptides, as confirmed by X‐ray photoelectron spectroscopy. Additionally, the developed system affords possibility for repeated cell seeding, proliferation, and release on previously used substrates without any additional pretreatment steps. This new approach represents an alternative to thermally triggered cell‐sheet harvesting platforms, offering possibility of capture and proliferation of various rare cell lines via appropriate selection of the cell‐adhesive ligand.
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    Photodynamic Eradication of Pseudomonas aeruginosa with Ru-Photosensitizers Encapsulated in Enzyme Degradable Nanocarriers
    The development of new approaches for the treatment of the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa was targeted by enhancing the effect of local antimicrobial photodynamic therapy (aPDT) using poly(ethylene glycol)-block-poly(lactic acid) (PEG114-block-PLAx) nanocarriers that were loaded with a ruthenium-based photosensitizer (PS). The action of tris(1,10-phenanthroline) ruthenium (II) bis(hexafluorophosphate) (RuPhen3) encapsulated in PEG114-block-PLAx micelles and vesicles was shown to result in an appreciable aPDT inactivation efficiency against planktonic Pseudomonas aeruginosa. In particular, the encapsulation of the PS, its release, and the efficiency of singlet oxygen (1O2) generation upon irradiation with blue light were studied spectroscopically. The antimicrobial effect was analyzed with two strains of Pseudomonas aeruginosa. Compared with PS-loaded micelles, formulations of the PS-loaded vesicles showed 10 times enhanced activity with a strong photodynamic inactivation effect of at least a 4.7 log reduction against both a Pseudomonas aeruginosa lab strain and a clinical isolate collected from the lung of a cystic fibrosis (CF) patient. This work lays the foundation for the targeted eradication of Pseudomonas aeruginosa using aPDT in various medical application areas.
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    Spectroscopic Investigation of the Quadruplex DNA‐Binding Properties of 9‐Aryl‐Substituted Isoquinolinium Derivatives
    The spectroscopic investigation of the binding properties of berberine‐type 9‐aryl‐substituted isoquinolinium derivatives with G‐quadruplex DNA (G4‐DNA) are presented. Photometric titrations show that these ligands bind with high affinity to the telomeric G4‐DNA form 22AG ( K =1.0–44×10 5 M −2 ). Furthermore, fluorimetric analysis of thermal DNA denaturation (FRET melting) reveals a significant thermal stabilization of G4‐DNA 22AG upon association with the methoxy‐substituted derivatives. As an analytically useful property, the derivatives with a phenyl substituent or with additional electron‐donating groups show a very weak fluorescence intensity, which increased significantly upon G4‐DNA complexation (fluorescence light‐up effect). Additional time‐resolved fluorescence spectroscopy indicated increased fluorescence lifetimes of the DNA‐bound 9‐(4‐methoxyphenyl)‐substituted derivative, when interacting with the quadruplex‐forming strand 22AG . Notably, the changes of the steady‐state and time‐resolved emission properties of the ligand are more pronounced with G4‐DNA than with duplex DNA so that the combination of these complementary methods may be used for the selective G4‐DNA detection.
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      2