Amphiphilic Polymer Hydrogel-supported Catalysts: Tuning the Accessibility to the Catalytic Site by Molecular Jacketing.

Crosslinked amphiphilic hydrogels, prepared using a peripherally clickable hyperbranched polyester (HBP) and PEG-diazides of different molecular weights, were used to ligate Cu utilizing the triazole rings formed by the alkyne-azide click reaction. Since only a fraction of the peripheral propargyl groups in the HB polyester are needed to generate the crosslinked polymer, the remaining were clicked with different types of azides, such as MPEG azide, decyl azide or 4-methylbenzyl azide, to create a molecular jacket around the catalytic sites that can potentially influence the catalytic activity and reaction outcome. The crosslinked films ligated with Cu functioned very effectively to catalyse alkyne-azide click reactions, both in water and in organic solvents; the nature of the molecular jacket around the catalytic site had a clear influence the reaction rate, which depended upon the relative solubilities of the reactants. The gel-supported catalyst films were reused multiple times with little loss in catalytic activity.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

New subnanomolar cathepsin S inhibitors with high selectivity: Optimizing covalent-reversible α -fluorovinylsulfones and -sulfonates as potential immunomodulators in cancer.

The cysteine protease cathepsin S (CatS) is overexpressed in many tumors. It is known to be involved in tumor progression as well as antigen processing in antigen-presenting cells (APC). Recent evidence suggests that silencing CatS improves the anti-tumor immune response in several cancers. Therefore, CatS is an interesting target to modulate the immune response in these diseases. Here, we present a series of covalent-reversible CatS inhibitors based on the α-fluorovinylsulfone and -sulfonate warheads. We optimized two lead structures by molecular docking approaches, resulting in 22 final compounds which were evaluated in fluorometric enzyme assays for CatS inhibition and for selectivity towards the off-targets CatB and CatL. The most potent inhibitor in the series has subnanomolar affinity (Ki = 0.08 nM) and more than 100,000-fold selectivity towards cathepsins B and L. These new reversible and non-cytotoxic inhibitors could serve as interesting leads to develop new immunomodulators in cancer therapy.

Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

Introducing carbohydrate patterning in mannose-decorated supramolecular assemblies and hydrogels.

Benzene-1,3,5-tricarboxamide (BTA) glyco-monomers containing one, two or three mannose units are synthesized and formulated into differently patterned supramolecular glycopolymers through homo-assembly or co-assembly with non-functionalized BTAs. Unfortunately, no cellular activity could be detected. Excitingly, these glyco-BTA monomers could be formulated into hydrogels, paving the way for (immune) cell culture.

Synthesis, radiolabeling, and preclinical in vivo evaluation of 68Ga-radiolabelled nanodiamonds.

PURPOSE: Nanodiamonds (NDs) represent a new class of nanoparticles and have gained increasing interest in medical applications. Modifying the surface coating by attaching binding ligands or imaging probes can transform NDs into multi-modal targeting probes. This study evaluated the biokinetics and biodistribution of 68Ga-radiolabelled NDs in a xenograft model. PROCEDURES: NDs were coated with an albumin-derived copolymer modified with desferrioxamine to provide a chelator for radiolabeling. In vivo studies were conducted in AR42J tumor-bearing CD1 mice to evaluate biodistribution and tumor accumulation of the NDs. RESULTS: Coated NDs were successfully radiolabeled using 68Ga at room temperature with radiolabeling efficiencies up to 91.8 ± 3.2 % as assessed by radio-TLC. In vivo studies revealed the highest accumulation in the liver and spleen, whereas tumor radioactivity concentration was low. CONCLUSIONS: Radiolabeling of coated NDs could be achieved. However, the obtained results indicate these coated NDs' limitations in their biodistribution within the conducted studies.

Preclinical PET and MR Evaluation of 89Zr- and 68Ga-Labeled Nanodiamonds in Mice over Different Time Scales.

Nanodiamonds (NDs) have high potential as a drug carrier and in combination with nitrogen vacancies (NV centers) for highly sensitive MR-imaging after hyperpolarization. However, little remains known about their physiological properties in vivo. PET imaging allows further evaluation due to its quantitative properties and high sensitivity. Thus, we aimed to create a preclinical platform for PET and MR evaluation of surface-modified NDs by radiolabeling with both short- and long-lived radiotracers. Serum albumin coated NDs, functionalized with PEG groups and the chelator deferoxamine, were labeled either with zirconium-89 or gallium-68. Their biodistribution was assessed in two different mouse strains. PET scans were performed at various time points up to 7 d after i.v. injection. Anatomical correlation was provided by additional MRI in a subset of animals. PET results were validated by ex vivo quantification of the excised organs using a gamma counter. Radiolabeled NDs accumulated rapidly in the liver and spleen with a slight increase over time, while rapid washout from the blood pool was observed. Significant differences between the investigated radionuclides were only observed for the spleen (1 h). In summary, we successfully created a preclinical PET and MR imaging platform for the evaluation of the biodistribution of NDs over different time scales.

Advanced EPI-X4 Derivatives Covalently Bind Human Serum Albumin Resulting in Prolonged Plasma Stability.

Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.

A Carbon Nanodot Based Near-Infrared Photosensitizer with a Protein-Ruthenium Shell for Low-Power Photodynamic Applications.

Near-infrared (NIR) light-activated photosensitization represents an encouraging therapeutic method in photodynamic therapy, especially for deep tissue penetration. In this context, two-photon activation, i.e., utilization of photons with relatively low energy but high photon flux for populating a virtual intermediate state leading to an excited state, is attractive. This concept would be highly advantageous in photodynamic therapy due to its minimal side effects. Herein, we propose that the combination of plasma protein serum albumin (HSA) containing several Ru complexes and NIR two-photon excitable carbon nanodots (Cdots), termed HSA-Ru-Cdots, provides several attractive features for enhancing singlet oxygen formation within the mitochondria of cancer cells stimulated by two-photon excitation in the NIR region. HSA-Ru-Cdot features biocompatibility, water solubility, and photostability as well as uptake into cancer cells with an endosomal release, which is an essential feature for subcellular targeting of mitochondria. The NIR two-photon excitation induced visible emission of the Cdots allows fluorescence resonance energy transfer (FRET) to excite the metal-to-ligand charge transfer of the Ru moiety, and fluorescence-lifetime imaging microscopy (FLIM) has been applied to demonstrate FRET within the cells. The NIR two-photon excitation is indirectly transferred to the Ru complexes, which leads to the production of singlet oxygen within the mitochondria of cancer cells. Consequently, we observe the destruction of filamentous mitochondrial structures into spheroid aggregates within various cancer cell lines. Cell death is induced by the long-wavelength NIR light irradiation at 810 nm with a low power density (7 mW/cm2), which could be attractive for phototherapy applications where deeper tissue penetration is crucial.

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.

A Polyclonal Selex Aptamer Library Directly Allows Specific Labelling of the Human Gut Bacterium Blautia producta without Isolating Individual Aptamers.

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome.

Light-Controlled Traceless Protein Labeling via Decaging Thio-o-naphthoquinone Methide Chemistry.

We report the molecular design of a novel multifunctional reagent and its application for light-controlled selective protein labeling. This molecule integrates functions of protein-ligand recognition, bioconjugation, ligand cleavage, and photoactivation by merging the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand and fluorescein. Highly electrophilic o-naphthoquinone methide was photochemically released and underwent proximity-driven selective labeling with the protein of interest (e.g., carbonic anhydrases), which retains its native function after labeling.

Polymer Chemistry in Living Cells.

ConspectusThe polymerization of biomolecules is a central operation in biology that connects molecular signals with proliferative and information-rich events in cells. As molecules arrange precisely across 3-D space, they create new functional capabilities such as catalysis and transport highways and exhibit new phase separation phenomena that fuel nonequilibrium dynamics in cells. Hence, the observed polymer chemistry manifests itself as a molecular basis leading to cellular phenotypes, expressed as a multitude of hierarchical structures found in cell biology. Although many milestone discoveries had accompanied the rise of the synthetic polymer era, fundamental studies were realized within a closed, pristine environment and that their behavior in a complex multicomponent system remains challenging and thus unexplored. From this perspective, there is a rich trove of undiscovered knowledge that awaits the polymer science community that can revolutionize understanding in the interactive nanoscale world of the living cell.In this Account, we discuss the strategies that have enabled synthetic polymer chemistry to be conducted within the cells (membrane inclusive) and to establish monomer design principles that offer spatiotemporal control of the polymerization. As reaction considerations such as monomer concentration, polymer growth dynamics, and reactivities are intertwined with the subcellular environment and transport processes, we first provide a chemical narrative of each major cellular compartment. The conditions within each compartment will therefore set the boundaries on the type of polymer chemistry that can be conducted. Both covalent and supramolecular polymerization concepts are explored separately in the context of scaffold design, polymerization mechanism, and activation. To facilitate transport into a localized subcellular space, we show that monomers can be reversibly modified by targeting groups or stimulus-responsive motifs that react within the specific compartment. Upon polymerization, we discuss the characterization of the resultant polymeric structures and how these phase-separated structures would impact biological processes such as cell cycle, metabolism, and apoptosis. As we begin to integrate cellular biochemistry with in situ polymer science, we identify landmark challenges and technological hurdles that, when overcome, would lead to invaluable discoveries in macromolecular therapeutics and biology.

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

Cyclic polymers: synthesis, characteristics, and emerging applications.

Cyclic polymers with a ring-like topology and no chain ends are a unique class of macromolecules. In the past several decades, significant advances have been made to prepare these fascinating polymers, which allow for the exploration of their topological effects and potential applications in various fields. In this Review, we first describe representative synthetic strategies for making cyclic polymers and their derivative topological polymers with more complex structures. Second, the unique physical properties and self-assembly behavior of cyclic polymers are discussed by comparing them with their linear analogues. Special attention is paid to highlight how polymeric rings can assemble into hierarchical macromolecular architectures. Subsequently, representative applications of cyclic polymers in different fields such as drug and gene delivery and surface functionalization are presented. Last, we envision the following key challenges and opportunities for cyclic polymers that may attract future attention: large-scale synthesis, efficient purification, programmable folding and assembly, and expansion of applications.

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

Combination of Six Individual Derivatives of the Pom-1 Antibiofilm Peptide Doubles Their Efficacy against Invasive and Multi-Resistant Clinical Isolates of the Pathogenic Yeast Candida albicans.

In previous studies, derivatives of the peptide Pom-1, which was originally extracted from the freshwater mollusk Pomacea poeyana, showed an exceptional ability to specifically inhibit biofilm formation of the laboratory strain ATCC 90028 as a model strain of the pathogenic yeast Candida albicans. In follow-up, here, we demonstrate that the derivatives Pom-1A to Pom-1F are also active against biofilms of invasive clinical C. albicans isolates, including strains resistant against fluconazole and/or amphotericin B. However, efficacy varied strongly between the isolates, as indicated by large deviations in the experiments. This lack of robustness could be efficiently bypassed by using mixtures of all peptides. These mixed peptide preparations were active against biofilm formation of all the isolates with uniform efficacies, and the total peptide concentration could be halved compared to the original MIC of the individual peptides (2.5 µg/mL). Moreover, mixing the individual peptides restored the antifungal effect of fluconazole against fluconazole-resistant isolates even at 50% of the standard therapeutic concentration. Without having elucidated the reason for these synergistic effects of the peptides yet, both the gain of efficacy and the considerable increase in efficiency by combining the peptides indicate that Pom-1 and its derivatives in suitable formulations may play an important role as new antibiofilm antimycotics in the fight against invasive clinical infections with (multi-) resistant C. albicans.

In Situ Assembly of Platinum(II)-Metallopeptide Nanostructures Disrupts Energy Homeostasis and Cellular Metabolism.

Nanostructure-based functions are omnipresent in nature and essential for the diversity of life. Unlike small molecules, which are often inhibitors of enzymes or biomimetics with established methods of elucidation, we show that functions of nanoscale structures in cells are complex and can implicate system-level effects such as the regulation of energy and redox homeostasis. Herein, we design a platinum(II)-containing tripeptide that assembles into intracellular fibrillar nanostructures upon molecular rearrangement in the presence of endogenous H2O2. The formed nanostructures blocked metabolic functions, including aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. As a consequence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated. We demonstrate that assembly-driven nanomaterials offer a rich avenue to achieve broad-spectrum bioactivities that could provide new opportunities in drug discovery.