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Hydrogel-based injectable drug delivery systems provide temporally and spatially controlled drug release with reduced adverse effects on healthy tissues. Therefore, they represent a promising therapeutic option for unresectable solid tumor entities. In this study, a peptide-starPEG/hyaluronic acid-based physical hydrogel is modified with ferrocene to provide a programmable drug release orchestrated by matrix-drug interaction and local reactive oxygen species (ROS). The injectable ROS-responsive hydrogel (hiROSponse) exhibits adequate biocompatibility and biodegradability, which are important for clinical applications. HiROSponse is loaded with the two cytostatic drugs (hiROSponsedox/ptx) doxorubicin (dox) and paclitaxel (ptx). Dox is a hydrophilic compound and its release is mainly controlled by Fickian diffusion, while the hydrophobic interactions between ptx and ferrocene can control its release and thus be regulated by the oxidation of ferrocene to the more hydrophilic state of ferrocenium. In a syngeneic malignant melanoma-bearing mouse model, hiROSponsedox/ptx slows tumor growth without causing adverse side effects and doubles the relative survival probability. Programmable release is further demonstrated in a tumor model with a low physiological ROS level, where dox release, low dose local irradiation, and the resulting ROS-triggered ptx release lead to tumor growth inhibition and increased survival.

summary over: (a) raw data for the metabolic assays (b) raw data for the analysis of the permeability of the capsules (c) unpublished images: typical cross section analysis of the capsules and organoids

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

Biomolecular and physical stimuli, such as stiffness and stress, of the extracellular environment, regulate collective cell dynamics and tissue patterning. The viscosity in the tumor microenvironment can increase due to the accumulation of macromolecules over time. Islands of rigid tumors are surrounded by soft cells that are more deformable than their healthy counterparts. Nonetheless, how the viscosities of the tumor microenvironment regulate collective cell spatial and temporal organization is not fully understood. Here, we used the human hepatoma (HepG2) cancer cells, the basic structural component of the liver, as an example to study the influence of viscosity (range from 0.8 cP to 15 cP) on cancer cell collective behavior in 3D microcapsules reactors. Alginate/Alginate-carboxymethylcellulose microcapsules (AL/AL-CMC MCs) with HepG2 cells were generated using a home-made high-throughput droplet-based microfluidic platform. Cell distribution, cell proliferation, spheroids growth, morphology change, and cytoskeleton difference were observed and quantified, showing a significant effect on viscosity change. Importantly, F-actin and keratin 8 intensity and distribution results can be a cue that viscosity increases enhancing the ability of cancer cells to squeeze through dense tissue. The results thus demonstrate that extracellular viscosity as an important physical cue regulates tumor development relevance to cancer biology.

The data concerns the preparation of two new 18F-labeled radiotracers using a ultra-fast radiolabeling method for tumor detection using positron emission tomography (PET) imaging.

The COVID-19 pandemic caused by SARS-CoV-2 led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options, which can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2 infected animal models, but also for (ii) clearance of virus infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

The selective inhibition of enzymes that catalyze the conversion of arachidonic acid to inflammatory eicosanoids represents a promising approach for cancer therapy. We, therefore, focus on the incorporation of metabolically stable, sterically demanding and hydrophobic carboranes into existing dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids. Here, we present the first carborane-containing dual COX-2/5-LO inhibitors derived from RWJ-63556. The replacement of the fluorophenyl moiety by meta- or para-carborane resulted in five carborane-containing derivatives 3, 6, 9, 13 and 17 that show high inhibitory activities toward COX-2 and 5-LO in vitro. Cell viability studies on the A375 melanoma cell line revealed that meta-carborane derivative 3 shows higher anticancer activity compared to RWJ-63556 based on accumulation of lipid droplets in the cells due to blockage of the COX-2 and 5-LO pathways, indicating a promising approach for the design of potent dual COX-2/5-LO inhibitors.

Fenoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) against rheumatoid arthritis, degenerative joint disease, ankylosing spondylitis and gout. Like other NSAIDs, fenoprofen inhibits the synthesis of prostaglandins by blocking both cyclooxygenase (COX) isoforms, COX-1 the “house-keeping” enzyme and COX-2 the induced isoform from pathological stimuli. Unselective inhibition of both COX isoforms results in many side effects, but off-target effects have also been reported. The steric modifications of the drugs could afford the desired COX-2 selectivity. Furthermore, NSAIDs have shown promising cytotoxic properties. The structural modification of fenoprofen using bulky dicarba-closo-dodecaborane(12) (carborane) clusters and the biological evaluation of the carborane analogues for COX inhibition and antitumor potential showed that the carborane analogues exhibit stronger antitumor potential compared to their respective aryl-based compounds.

Detection of antigens and antibodies (Abs) is of great importance in determining the infection and immunity status of the population, as they are key parameters guiding the handling of pandemics. Current point-of-care (POC) devices are a convenient option for rapid screening; however, their sensitivity requires further improvement. We present an interdigitated gold nanowire-based impedance nanobiosensor to detect COVID-19-associated antigens (receptor-binding domain of S1 protein of the SARS-CoV-2 virus) and respective Abs appearing during and after infection. The electrochemical impedance spectroscopy technique was used to assess the changes in measured impedance resulting from the binding of respective analytes to the surface of the chip. After 20 min of incubation, the sensor devices demonstrate a high sensitivity of about 57 pS·sn per concentration decade and a limit of detection (LOD) of 0.99 pg/mL for anti-SARS-CoV-2 Abs and a sensitivity of around 21 pS·sn per concentration decade and an LOD of 0.14 pg/mL for the virus antigen detection. Finally, the analysis of clinical plasma samples demonstrates the applicability of the developed platform to assist clinicians and authorities in determining the infection or immunity status of the patients.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used therapeutics against pain, fever and inflammation; additionally, antitumor properties have been reported. NSAIDs reduce the synthesis of prostaglandins (PG) by inhibiting the cyclooxygenase (COX) isoforms COX-1 and COX-2. As non-selective inhibition is associated with off-target effects, strategies to achieve selectivity for the clinically preferred isoform COX-2 are of high interest. The modification of NSAIDs using carborane clusters as phenyl mimetics has been reported to alter the selectivity profile through size exclusion. Inspired by these findings, we have prepared isonimesulide and its carborane derivatives. The biological screening showed that the carborane containing compounds exhibit a stronger antitumor potential compared to nimesulide and isonimesulide. Furthermore, the replacement of the phenyl ring of isonimesulide with a carborane moiety resulted in a shift of the COX activity from non-active to COX-active compounds.

Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that show no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibit lower anticancer activity compared to the related di-tert-butylphenols. Inter-estingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell pro-liferation more potently than its carbon-based R-830 counterpart. Considering all the ad-vantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.

We demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) spheroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules was selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer and/or filtering of the biochemical species. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Metabolic activity of spheroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Because of the facile tuning of the shell thickness and permeability, we believe that our system is suitable for studying the formation of cancer spheroids and their functional interaction with the surrounding microenvironment.

Multi-target drugs (MTDs) are emerging alternatives to combination therapies. Since both his-tone deacetylases (HDACs) and cyclooxygenase-2 (COX-2) are known to be overexpressed in several cancer types, we herein report the design, synthesis, and biological evaluation of a li-brary of dual HDAC-COX inhibitors. The designed compounds were synthesized via an efficient parallel synthesis approach using preloaded solid-phase resins. Biological in vitro assays demon-strated that several of the synthesized compounds possess pronounced inhibitory activities against HDAC and COX isoforms. The membrane permeability and inhibition of cellular HDAC activity of selected compounds were confirmed by whole-cell HDAC inhibition assays and western blot experiments. The most promising dual inhibitors C3 and C4 evoked antiprolifera-tive effects in the low micromolar concentration range and caused a significant increase in apoptotic cells. In contrast to previous reports, the simultaneous inhibition of HDAC and COX activity by dual HDAC-COX inhibitors or combination treatment with vorinostat and celecoxib did not result in additive or synergistic anticancer activities.

The presence of inflammatory mediators in the tumor microenvironment, such as cytokines, growth factors or eicosanoids, indicate cancer-related inflammatory processes. Targeting these inflammatory mediators and related signal pathways may offer a rational strategy for the treatment of cancer. This study focuses on the incorporation of metabolically stable, sterically demanding, and hydrophobic dicarba-closo-dodecaboranes (carboranes) into dual cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids. The di-tert-butylphenol derivative tebufelone represents a selective dual COX-2/5-LO inhibitor. The incorporation of meta- or para-carborane into the tebufelone scaffold resulted in eight carborane-based tebufelone analogs that show no COX inhibition but 5-LO inhibitory activities in vitro. Cell viability studies on HT29 colon adenocarcinoma cells revealed that the para-carborane analog 8 exhibits higher anticancer activity compared to tebufelone through the inhibition of cell proliferation. Hence, this strategy proved to be a promising approach to design potent 5-LO inhibitors with potential application as cytostatic agents.

Biomolecular and physical stimuli, such as stiffness and stress, of the extracellular environment, regulate collective cell dynamics and tissue patterning. The viscosity in the tumor microenvironment can increase due to the accumulation of macromolecules over time. Islands of rigid tumors are surrounded by soft cells that are more deformable than their healthy counterparts. Nonetheless, how the viscosities of the tumor microenvironment regulate collective cell spatial and temporal organization is not fully understood. Here, we used the human hepatoma (HepG2) cancer cells, the basic structural component of the liver, as an example to study the influence of viscosity (range from 0.8 cP to 15 cP) on cancer cell collective behavior in 3D microcapsules reactors. Alginate/Alginate-carboxymethylcellulose microcapsules (AL/AL-CMC MCs) with HepG2 cells were generated using a home-made high-throughput droplet-based microfluidic platform. Cell distribution, cell proliferation, spheroids growth, morphology change, and cytoskeleton difference were observed and quantified, showing a significant effect on viscosity change. Importantly, F-actin and keratin 8 intensity and distribution results can be a cue that viscosity increases enhancing the ability of cancer cells to squeeze through dense tissue. The results thus demonstrate that extracellular viscosity as an important physical cue regulates tumor development relevance to cancer biology.

This publication includes datasets of electrical impedance spectroscopy measurements of the response of functionalized gold nanowires when interacting with antigens and antibodies related to SARS-CoV-2 in physiological conditions and in human plasma samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic with millions of infections and deaths worldwide and devastating impact on global economy. Up to now, vaccines and monoclonal antibody (mAb) therapies lack to provide a long-lasting protection against rapidly evolving new emerging SARS-CoV-2 variants. Thus, novel therapeutic options are pressingly needed especially for immunocompromised patients and/or patients with high risk for developing a severe coronavirus disease 2019 (COVID-19). In that regard, we developed a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2). This ACE2 decoy potently binds to the SARS-CoV-2 receptor binding domain (RBD), neutralizes SARS-CoV-2 as well as the Delta and Omicron variant and protects hamsters from a SARS-CoV-2 infection. To additionally use this ACE2 decoy for elimination of virus infected cells, we equipped it with an epitope tag. Thus, it can be applied as adapter molecule in the modular platform technologies UniMAB and UniCAR, which already demonstrated great success in the setting of malignant diseases. As adapter molecule the ACE2 decoy is able to efficiently recruit either universal chimeric antigen receptor (UniCAR) modified T cells or, in combination with an anti-peptide epitope-anti-CD3 bispecific Ab of the UniMAB system, unmodified T cells to efficiently kill SARS-CoV-2 RBD expressing human cells. Taken together, the ACE2 decoy represents a very promising immunotherapeutic drug for both SARS-CoV-2 variant neutralization and infected cell killing via the UniMAB and UniCAR system and might, therefore, clearly improve the treatment of COVID-19 patients.

Liver cancer had the fastest increasing mortality for decades. Compared to 2D models, that cannot simulate the microenvironment, and key functions of organs, 3D organoids models are now entering the field of in vitro analysis. Here, alginate-chitosan hybrid microcapsules are fabricated with a high reproducible cross junction based microfluidic droplet generation system. With high reproducibility and compartmentalization, 103 of microcapsules can be generated within a few minutes. The effects of fluid flow rates on its sizes and shell thicknesses were systematically studied, forming microcapsules with different capsule diameters (~ 300 to 700 µm) and thicknesses (~ 5 to 150 µm). The semi-permeability of these capsules has been well studied, combined a COMSOL model. These microcapsules, with a suitable diffusion rate for nutrients, are applied for human hepatoma cell line encapsulation. Metabolic of organoids in capsules are confirmed by tracking substrates (testosterone) and metabolites (androstenedione). Overall, highly repeatable alginate-chitosan hybrid microcapsules obtained by microfluidic droplet method are not only suitable for liver cancer cells culturing, but also promising for various cell organoids generation.

Liver cancer had the fastest increasing mortality for decades. Compared to 2D models, that cannot simulate the microenvironment, and key functions of organs, 3D organoids models are now entering the field of in vitro analysis. Here, alginate-chitosan hybrid microcapsules are fabricated with a high reproducible cross junction based microfluidic droplet generation system. With high reproducibility and compartmentalization, 103 of microcapsules can be generated within a few minutes. The effects of fluid flow rates on its sizes and shell thicknesses were systematically studied, forming microcapsules with different capsule diameters (~ 300 to 700 µm) and thicknesses (~ 5 to 150 µm). The semi-permeability of these capsules has been well studied, combined a COMSOL model. These microcapsules, with a suitable diffusion rate for nutrients, are applied for human hepatoma cell line encapsulation. Metabolic of organoids in capsules are confirmed by tracking substrates (testosterone) and metabolites (androstenedione). Overall, highly repeatable alginate-chitosan hybrid microcapsules obtained by microfluidic droplet method are not only suitable for liver cancer cells culturing, but also promising for various cell organoids generation.

Background: Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest in the development of numerous in vitro and in vivo tumor models. Study Aims: To study the influence of different permeabilities of microcapsules (MCs) on different cancer cell proliferations, and to design and engineer the formation of 3D tumor clusters in MCs. Materials and Methods: A fluidics-based low-cost methodology was used to reproducibly generate alginate (AL) and alginate-chitosan (AL-CS) MCs in a cross-junctions water-in-oil system. The diffusion through the shell of AL and AL-CS MCs was monitored using fluorescein sodium (376 Da), FITC-Dextran 70 (70 kDa), and FITC-Dextran 2000 (2000 kDa) as fluorescent probes representing small molecules, proteins, and macromolecules, respectively. HepG2 Red FLuc (human hepatoma cell line) and A375 (human melanoma cell line) cultured in high-glucose DMEM medium were used to study the proliferation differences in terms of dimensions and geometries in AL and AL-CS MCs. The metabolic activity of tumor clusters in MCs was confirmed by tracking the turnover of testosterone to androstenedione with lower case Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: HepG2 Red FLuc and A375 cells show different proliferation properties in AL and AL-CS MCs. A375 tumor clusters grow faster in more permeable AL MCs and slower in less permeable AL-CS MCs. In the case of HepG2 Red FLuc, a significant difference in proliferation rate was not observed between AL and AL-CS MCs at the early stage (1 week). Interestingly, it was observed that different loose and tight cell cluster morphologies can form, also including cell proliferation along radial directions in both MC types and both cell lines. Cytochrome P450 (CYP)-dependent metabolization of testosterone by both HepG2 Red FLuc and A375 tumor clusters in the AL and AL-CS MCs showed the same trends in good

The development of point-of-care (POC) testing platforms has increased during the COVID-19 pandemic due to their multiple benefits including low cost, rapid turnaround time, on-site testing, and minimal sample preparation [1-2]. Although POC tests are a good alternative to the gold standard technique (reverse-transcriptase-polymerase chain reaction, RT-PCR) for SARS-CoV-2 detection, there are challenges regarding their sensitivity and specificity that need to be addressed [3-4]. One strategy to improve the performance of POC is the integration of nanostructures as sensing elements [5]. In this work, we used interdigitated gold nanowires (Au NWs) in combination with electrical impedance spectroscopy (EIS) for the detection of the receptor-binding domain of the S1 protein of the SARS-CoV-2 virus and the respective antibodies that appear during and after infection. Our sensor system was composed of six sensing devices, each of these sensors containing six pairs of interdigitated gold nanowires of 120 nm in width. The surface of the Au NWs was functionalized with antigens or antibodies of SARS-CoV-2 so that the molecules of interest present in the sample can bind to them. The adhesion of molecules to the surface of the Au NWs modulates the physicochemical properties of the surface [6]. As a result, it was possible to correlate the changes in electrical impedance with the binding of specific analytes to the surface of the Au NWs using EIS. The developed sensing platform is an attractive system for screening during pandemics and can be adapted for the detection of relevant target-analyte pairs in different diseases. References [1] E. Valera et al., ?COVID-19 Point-of-Care Diagnostics: Present and Future,? ACS Nano, vol. 15, no. 5, pp. 7899?7906, 2021, doi: 10.1021/acsnano.1c02981. [2] E. Morales-Narváez and C. Dincer, ?The impact of biosensing in a pandemic outbreak: COVID-19,? Biosens. Bioelectron., vol. 163, p. 112274, 2020, doi: https://doi.org/10.1016/j.bios.2020.1122

Liver cancer is the second most lethal malignancy worldwide. Recently, three-dimensional (3D) cancer organoids models have been constructed and applied to liver cancer research, to predict the therapy outcomes. However, there is still low success and reproducibility rate for generating patient-derived liver tumoroids due to limitations in current technologies. Herein, a high reproducible cross junction based microfluidic droplet generation system is applied for human hepatoma cell line encapsulation and organoids engineering. The fabrication of alginate and alginate-chitosan microcapsules is systematically studied, forming microcapsules with different shell thicknesses (~ 5 to 150 µm) and tunable permeability. In combination with a COMSOL model, the size selective permeability of different molecular complexes through the capsule membrane has been investigated, which is essential to ensure efficient mass transfer of small molecules, and prevent large substances from reaching the loaded cells. Finally, we demonstrate that the cells are prone to aggregate more tightly in capsules with a lower permeability, which causing more hypotonicity and lower viability. Because of the high reproducibility, compartmentalization, and easily permeability tuning, this system not only provides a great platform for liver patient-derived tumoroids forming, but also is promising for other cell organoids design and engineering.

The characterization of novel radiotracers toward their metabolic stability is an essential part for their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wild-type HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio-thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement to the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

1-(4-Fluorobenzoyl)-9H-carbazole (1) was synthesized starting from 9H carbazole and 4-fluorobenzonitrile by Friedel-Crafts acylation using boron trichloride to direct the substitu-tion in 1-position. Single X-ray crystal structure analysis unambiguously revealed the molecular structure of 1.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tu-mor microenvironment, tumor growth, apoptosis resistance, angiogenesis, and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the effect of functional knockout of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by western blot and flow cytometry analysis. When comparing COX-2 knockout cells to con-trols, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor devel-opment in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic ac-tivity of cells under normoxia and experimental hypoxia as demonstrated by using the radio-tracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.

Mefenamic acid represents a widely used nonsteroidal anti-inflammatory drug (NSAID) to treat pain of postoperative sur-gery and heavy menstrual bleeding. Like other NSAIDs, mefenamic acid inhibits the synthesis of prostaglandins by non-selectively blocking cyclooxygenase (COX) isoforms COX-1 and COX-2. For improved selectivity of the drug and thereby reduced related side effects, the carborane analogues of mefenamic acid were evaluated. The ortho-, meta- and para-carborane derivatives were synthesized in three steps: halogenation of the respective cluster, followed by a Pd-catalyzed B–N coupling and hydrolysis of the nitrile derivatives under acidic conditions. COX inhibitory activity and cytotoxicity for different cancer cell lines revealed that the carborane analogues have stronger anti-tumor potential compared to their parent organic compound.

Liver cancer is the second most lethal malignancy worldwide. Recently, three-dimensional (3D) cancer organoids models have been constructed and applied to liver cancer research, to predict the therapy outcomes. However, there is still low success and reproducibility rate for generating patient-derived liver tumoroids due to limitations in current technologies. Herein, a high reproducible cross junction based microfluidic droplet generation system is applied for human hepatoma cell line encapsulation and organoids engineering. The fabrication of alginate and alginate-chitosan microcapsules is systematically studied, forming microcapsules with different shell thicknesses (~ 5 to 150 µm) and tunable permeability. In combination with a COMSOL model, the size selective permeability of different molecular complexes through the capsule membrane has been investigated, which is essential to ensure efficient mass transfer of small molecules, and prevent large substances from reaching the loaded cells. Finally, we demonstrate that the cells are prone to aggregate more tightly in capsules with a lower permeability, which causing more hypotonicity and lower viability. Because of the high reproducibility, compartmentalization, and easily permeability tuning, this system not only provides a great platform for liver patient-derived tumoroids forming, but also is promising for other cell organoids design and engineering.

Background: Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest in the development of numerous in vitro and in vivo tumor models. Study Aims: To study the influence of different permeabilities of microcapsules (MCs) on different cancer cell proliferations, and to design and engineer the formation of 3D tumor clusters in MCs. Materials and Methods: A fluidics-based low-cost methodology was used to reproducibly generate alginate (AL) and alginate-chitosan (AL-CS) MCs in a cross-junctions water-in-oil system. The diffusion through the shell of AL and AL-CS MCs was monitored using fluorescein sodium (376 Da), FITC-Dextran 70 (70 kDa), and FITC-Dextran 2000 (2000 kDa) as fluorescent probes representing small molecules, proteins, and macromolecules, respectively. HepG2 Red FLuc (human hepatoma cell line) and A375 (human melanoma cell line) cultured in high-glucose DMEM medium were used to study the proliferation differences in terms of dimensions and geometries in AL and AL-CS MCs. The metabolic activity of tumor clusters in MCs was confirmed by tracking the turnover of testosterone to androstenedione with lower case Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: HepG2 Red FLuc and A375 cells show different proliferation properties in AL and AL-CS MCs. A375 tumor clusters grow faster in more permeable AL MCs and slower in less permeable AL-CS MCs. In the case of HepG2 Red FLuc, a significant difference in proliferation rate was not observed between AL and AL-CS MCs at the early stage (1 week). Interestingly, it was observed that different loose and tight cell cluster morphologies can form, also including cell proliferation along radial directions in both MC types and both cell lines. Cytochrome P450 (CYP)-dependent metabolization of testosterone by both HepG2 Red FLuc and A375 tumor clusters in the AL and AL-CS MCs showed the same trends in good

The development of point-of-care (POC) testing platforms has increased during the COVID-19 pandemic due to their multiple benefits including low cost, rapid turnaround time, on-site testing, and minimal sample preparation [1-2]. Although POC tests are a good alternative to the gold standard technique (reverse-transcriptase-polymerase chain reaction, RT-PCR) for SARS-CoV-2 detection, there are challenges regarding their sensitivity and specificity that need to be addressed [3-4]. One strategy to improve the performance of POC is the integration of nanostructures as sensing elements [5]. In this work, we used interdigitated gold nanowires (Au NWs) in combination with electrical impedance spectroscopy (EIS) for the detection of the receptor-binding domain of the S1 protein of the SARS-CoV-2 virus and the respective antibodies that appear during and after infection. Our sensor system was composed of six sensing devices, each of these sensors containing six pairs of interdigitated gold nanowires of 120 nm in width. The surface of the Au NWs was functionalized with antigens or antibodies of SARS-CoV-2 so that the molecules of interest present in the sample can bind to them. The adhesion of molecules to the surface of the Au NWs modulates the physicochemical properties of the surface [6]. As a result, it was possible to correlate the changes in electrical impedance with the binding of specific analytes to the surface of the Au NWs using EIS. The developed sensing platform is an attractive system for screening during pandemics and can be adapted for the detection of relevant target-analyte pairs in different diseases. References [1] E. Valera et al., ?COVID-19 Point-of-Care Diagnostics: Present and Future,? ACS Nano, vol. 15, no. 5, pp. 7899?7906, 2021, doi: 10.1021/acsnano.1c02981. [2] E. Morales-Narváez and C. Dincer, ?The impact of biosensing in a pandemic outbreak: COVID-19,? Biosens. Bioelectron., vol. 163, p. 112274, 2020, doi: https://doi.org/10.1016/j.bios.2020.1122

Objectives A recently developed synthetic route for the transition metal-free reductive coupling of aryl boronic acids with nitro1 or nitroso2 substituted arenes gives access to a variety of diaryl amines like fenamates.3 Radiotracers targeting COX-2 have been developed and tested as PET tracer but no clinically approved radiotracer emerged up to now. Aim of this project is to evaluate this novel synthetic route as a general access to 18F-labeled N,N-diaryl amines which are principally not activated for 18F-labeling by nucleophilic aromatic substitution, and by that access to fenamates like flufenamic acid as COX-targeting radiotracers in a wider sense. In this report, we present first results with focus on radiolabeling and reactivity of an asymmetric 18F-labeled nitro-arene. Methods The asymmetric sydnone-substituted nitroarene was radiolabeled under optimized conditions with fluorine-18 using K222/K2CO3 in DMF at 90°C and was purified by a C18-based solid phase extraction (SPE). Elution from the dried C18 SPE-cartridge with ortho-dichlorobenzene over a SEP-Pak Dry cartridge provided the intermediate [18F]A for further testing and optimization of the reductive coupling step. Subsequent radiochemical conversion of [18F]A with different boronic acids was monitored via radio-UHPLC. 3-Cyanophenyl boronic acid was used to optimize the coupling reaction with [18F]A with respect to the parameters base/reducing agent, temperature, reaction time. Furthermore, [18F]A was subjected to the reaction with other ortho-, meta-, and para-substituted boronic acids to get a first impression about the scope of this reaction. Results [18F]A was isolated after radiolabeling and purification by SPE in 16-54% isolated RCY (n=4). Optimizations using aliquots of 50 µL allowed for optimization of several reaction conditions with one batch of [18F]A. Triphenylphosphine but not triethylphosphite was found to mediate the reductive coupling of 3-cyanophenylboronic acid at reaction temperatures

In the current COVID-19 pandemic that we are facing, it has become evident that the development of biosensors that can aid in the different stages of screening and surveillance: infection, disease progression and recovery, is extremely important. We developed a system that can be used for the detection of SARS-CoV-2 antibodies that are present during and after the infection. Our sensor chips were composed of six sensing devices, each of these containing interdigitated gold nanowires. The surfaces of the mentioned nanowires were functionalized with proteins of the SARS-CoV-2 so that the antibodies present in solution can bind to them. The detection of such antigen-antibody binding events was performed using the impedance spectroscopy technique. For this purposes, an AC signal over a range of frequencies (20 Hz-1 MHz) was applied to the sensor chips and the changes in impedance and phase were measured. The change in the overall impedance of the system was correlated to antigen-antibody binding events. The developed sensing system is a versatile platform that could be easily adapted to detect relevant target-analyte pairs in different diseases.

Objectives: The combination of non-invasive molecular imaging (PET/SPECT)- and optical imaging (OI) techniques for tumor identification and resection are emerging. This powerful strategy promises to precisely differentiate between healthy and affected tumor tissues which is of most relevance for preoperative planning (prestaging) followed by R0-tumor resection via image-guided intraoperative surgery. The goal of this work is the development of radiolabeled near-infrared (NIR) fluorophores for PET/SPECT and optical imaging. The fluorophores were prepared for radiolabeling with the positron emitter fluorine-18 and with the gamma emitter iodine-123 for SPECT imaging with the aim to elucidate their potential as imaging agents for the detection of tumor tissues. Moreover, the radiolabeled dyes are intended to be bioconjugated to the PSMA-1007 binding motif, as a generic prominent tumor targeting vector for enrichment in prostate tumors (1). Methods: We have developed fluorophores belonging to the silicon rhodamine (SiR) family with optical properties in the NIR spectral range (2). The photostable fluorophores have been characterized using NMR-, UV/VIS/NIR-spectroscopy and mass spectrometry. Furthermore the SiRs were radiolabeled by using the approach for copper-mediated radiolabeling of arylboronic acids, functioning as precursors both for fluorine-18 and iodine-123 labeling (3). The radiolabeling conditions were optimized based on radiochemical conversions (RCC) as analyzed by using radio-HPLC and radio-TLC. The model and lead compound [18F]F-SiR was isolated by radio-HPLC followed by SPE for further in vitro experiments, e.g. in vitro stability was determined in human serum. Results: Novel boronic acid functionalized SiRs with chemical yields up to 68% were received via multistep organic syntheses. The blue dyes show high extinction coefficients up to 95.000 M-1cm-1, quantum yields of 0.33 and high photo stability making them useful for NIR optical imaging. After caref

Objectives: Finding optimal 18F-fluorination conditions represents a critical and often time-consuming step in radiotracer development. Microfluidic or lab-on-a-chip devices are modern tools for that purpose but not accessible everywhere. Inspired by 18F-chemistry in low volumes based on a MAX/MCX-trapping technique for one complete 18F-batch,[1] we herein present an 18F-labeling approach using low volume aliquots (<100 µL) of defined QMA-eluates in HPLC vials as reaction vessels for optimizations as well as the preparation of radiotracers for preclinical studies. Methods: For optimization experiments, 1-80 MBq [18F]fluoride was eluted from QMA cartridges with defined mixtures of phase transfer catalyst (Kryptofix®222), base (such as K2CO3, KHCO3, KH2PO4), and 2-3% water in acetonitrile[2]. 25 or 50 µL aliquots of the eluates were pipetted into 1.5 mL HPLC vials and the sealed vials were consecutively dried once at 90°C for 3 min with a helium stream. After processing up to 15 vials in one batch, 25 or 50 µL precursor stock solution (1-20 mg/mL in MeCN, DMF, or DMSO) was added and the sealed vials were heated at defined temperatures (60-140°C) and times (5-15 min) by using three heaters equipped with aluminum blocks. Samples were 4-fold diluted with acetonitrile/water 50/50 for analysis by radio-TLC/radio-HPLC. 18F-trapping/elution at higher activity levels (~20 GBq [18F]fluoride) was performed by using a radiosynthesizer (Tracerlab FXFN) followed by transfer into a lead container having three small bores as inlet, vent needle and withdrawal port. A self-made pipet tip-to-cannula adapter allowed withdrawal of defined 25-100 µL aliquots at an activity level of ~13 MBq/µL for further processing under optimized conditions as described above. Results: The use of small reaction volumes and HPLC vials enabled an efficient 18F-optimization workflow to examine rapidly a variety of reaction parameters (>50 reactions/day) using minimal amounts of precursor. Utilizin

Objectives A recently developed synthetic route for the transition metal-free reductive coupling of aryl boronic acids with nitro1 or nitroso2 substituted arenes gives access to a variety of diaryl amines like fenamates.3 Radiotracers targeting COX-2 have been developed and tested as PET tracer but no clinically approved radiotracer emerged up to now. Aim of this project is to evaluate this novel synthetic route as a general access to 18F-labeled N,N-diaryl amines which are principally not activated for 18F-labeling by nucleophilic aromatic substitution, and by that access to fenamates like flufenamic acid as COX-targeting radiotracers in a wider sense. In this report, we present first results with focus on radiolabeling and reactivity of an asymmetric 18F-labeled nitro-arene. Methods The asymmetric sydnone-substituted nitroarene was radiolabeled under optimized conditions with fluorine-18 using K222/K2CO3 in DMF at 90°C and was purified by a C18-based solid phase extraction (SPE). Elution from the dried C18 SPE-cartridge with ortho-dichlorobenzene over a SEP-Pak Dry cartridge provided the intermediate [18F]A for further testing and optimization of the reductive coupling step. Subsequent radiochemical conversion of [18F]A with different boronic acids was monitored via radio-UHPLC. 3-Cyanophenyl boronic acid was used to optimize the coupling reaction with [18F]A with respect to the parameters base/reducing agent, temperature, reaction time. Furthermore, [18F]A was subjected to the reaction with other ortho-, meta-, and para-substituted boronic acids to get a first impression about the scope of this reaction. Results [18F]A was isolated after radiolabeling and purification by SPE in 16-54% isolated RCY (n=4). Optimizations using aliquots of 50 µL allowed for optimization of several reaction conditions with one batch of [18F]A. Triphenylphosphine but not triethylphosphite was found to mediate the reductive coupling of 3-cyanophenylboronic acid at reaction temperatures

Aim/Introduction: In recent years quinoline-based small molecules targeting the fibroblast activation protein alpha (FAP) have gained interest for imaging a variety of tumor entities (1). A number of radiotracers for SPECT and PET have been developed, but so far only three FAP inhibitor (FAPI) radioligands have been reported to be radiolabeled with fluorine-18 (2,3,4). This study shows the optimization and automation of the radiofluorination of FAPI-74. Materials and Methods: (S)-(4-Carboxymethyl-7-{2-[4-(3-{4-[2-(2-cyano-pyrrolidin-1-yl)-2-oxoethylcarbamoyl]-quinolin-6-yloxy}-propyl)-piperazin-1-yl]-2-oxo-ethyl}-[1,4,7]triazonan-1-yl)-acetic acid, commonly referred to as FAPI-74, was radiolabeled using the [18F]AlF chelation method. Starting from [18F]fluoride, the reaction with AlCl3, chelate formation and subsequent purification was initially optimized by manual syntheses. Optimization included the examination of different anion exchangers (QMA light, PSHCO3) and elution solutions (NaOAc buffer pH4, 0.9% NaCl) as well as careful adjustment of the reaction parameters time (0-20 min), temperature (r.t. to 100°C), amount of AlCl3 and NaOAc buffer pH4, solvents (DMSO, EtOH), and precursor concentration (1-350 µM). Radiochemical conversion (RCC) was determined by radio-UPLC of the crude reaction mixtures. Selected reaction mixtures were analyzed after decay using UPLC-MS to identify non-radioactive byproducts. The optimized radiosynthetic procedure was transferred to a fully automated radiosynthesizer (TRACERlab FXFN) and the final product was purified and formulated using semi-preparative HPLC and SPE. Results: Under optimized conditions, RCC of [18F]AlF-FAPI-74 of > 99% was still observed at precursor concentrations as low as 12 µM FAPI-74 after reaction in a 1:1 molar ratio with AlCl3 in DMSO/sodium acetate buffer at pH 4 at 80°C for 15 minutes. Transfer of optimized conditions and upscaling was successfully achieved and delivered radiochemica

Radiolabeled fluorescent dyes are decisive for bimodal imaging as well as highly in demand for nuclear- and optical imaging. Silicon-rhodamines (SiRs) show unique near-infrared (NIR) optical properties, large quantum yields and extinction coefficients as well as high photostability. Here, we describe the synthesis, characterization and radiolabeling of novel NIR absorbing and emitting fluorophores from the silicon-rhodamine family for use in optical imaging (OI) combined with positron emission tomography (PET) or single photon emission computed tomography (SPECT), respectively. The presented photostable SiRs were characterized using NMR-, UV-Vis-NIR-spectroscopy and mass spectrometry. Moreover, the radiolabeling conditions using fluorine-18 or iodine-123 were extensively explored. After optimization, the radiofluorinated NIR imaging agents were obtained with radiochemical conversions (RCC) up to 70% and isolated radiochemical yields (RCY) up to 54% at molar activities of g.t. 70 GBq/µmol. Radioiodination delivered RCCs over 92% and allowed to isolate the 123I-labeled product in RCY of 54% at a molar activity of g.t. 7.6 TBq/µmol. The radiofluorinated SiRs exhibit in vitro stabilities g.t. 70% after two hours in human serum. The first described radiolabeled SiRs are a promising step toward their further development as multimodal PET/SPECT-NIR imaging agents for planning and subsequent imaging-guided oncological surgery.

Since the first description of the nuclear autoantigens in the late sixties and early seventies of the last century we and many other groups have tried with difficulty to establish monoclonal anti-bodies (mabs) against nuclear antigens including to the autoantigen La/SS-B. To date, only a few anti-La mabs have been derived by conventional hybridoma technology. However, these pre-viously described anti-La mabs are not bona fide autoantibodies as they recognize either human La specific- or cryptic or post-translationally modified epitopes which are not accessible on na-tive mouse La protein. Herein we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adop-tive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermuta-tions occurring during T cell dependent maturation can lead to autoreactivity to the nuclear au-toantigen La/SS-B.

Objectives: The combination of non-invasive molecular imaging (PET/SPECT)- and optical imaging (OI) techniques for tumor identification and resection are emerging. This powerful strategy promises to precisely differentiate between healthy and affected tumor tissues which is of most relevance for preoperative planning (prestaging) followed by R0-tumor resection via image-guided intraoperative surgery. The goal of this work is the development of radiolabeled near-infrared (NIR) fluorophores for PET/SPECT and optical imaging. The fluorophores were prepared for radiolabeling with the positron emitter fluorine-18 and with the gamma emitter iodine-123 for SPECT imaging with the aim to elucidate their potential as imaging agents for the detection of tumor tissues. Moreover, the radiolabeled dyes are intended to be bioconjugated to the PSMA-1007 binding motif, as a generic prominent tumor targeting vector for enrichment in prostate tumors (1). Methods: We have developed fluorophores belonging to the silicon rhodamine (SiR) family with optical properties in the NIR spectral range (2). The photostable fluorophores have been characterized using NMR-, UV/VIS/NIR-spectroscopy and mass spectrometry. Furthermore the SiRs were radiolabeled by using the approach for copper-mediated radiolabeling of arylboronic acids, functioning as precursors both for fluorine-18 and iodine-123 labeling (3). The radiolabeling conditions were optimized based on radiochemical conversions (RCC) as analyzed by using radio-HPLC and radio-TLC. The model and lead compound [18F]F-SiR was isolated by radio-HPLC followed by SPE for further in vitro experiments, e.g. in vitro stability was determined in human serum. Results: Novel boronic acid functionalized SiRs with chemical yields up to 68% were received via multistep organic syntheses. The blue dyes show high extinction coefficients up to 95.000 M-1cm-1, quantum yields of 0.33 and high photo stability making them useful for NIR optical imaging. After caref

Objectives: Finding optimal 18F-fluorination conditions represents a critical and often time-consuming step in radiotracer development. Microfluidic or lab-on-a-chip devices are modern tools for that purpose but not accessible everywhere. Inspired by 18F-chemistry in low volumes based on a MAX/MCX-trapping technique for one complete 18F-batch,[1] we herein present an 18F-labeling approach using low volume aliquots (<100 µL) of defined QMA-eluates in HPLC vials as reaction vessels for optimizations as well as the preparation of radiotracers for preclinical studies. Methods: For optimization experiments, 1-80 MBq [18F]fluoride was eluted from QMA cartridges with defined mixtures of phase transfer catalyst (Kryptofix®222), base (such as K2CO3, KHCO3, KH2PO4), and 2-3% water in acetonitrile[2]. 25 or 50 µL aliquots of the eluates were pipetted into 1.5 mL HPLC vials and the sealed vials were consecutively dried once at 90°C for 3 min with a helium stream. After processing up to 15 vials in one batch, 25 or 50 µL precursor stock solution (1-20 mg/mL in MeCN, DMF, or DMSO) was added and the sealed vials were heated at defined temperatures (60-140°C) and times (5-15 min) by using three heaters equipped with aluminum blocks. Samples were 4-fold diluted with acetonitrile/water 50/50 for analysis by radio-TLC/radio-HPLC. 18F-trapping/elution at higher activity levels (~20 GBq [18F]fluoride) was performed by using a radiosynthesizer (Tracerlab FXFN) followed by transfer into a lead container having three small bores as inlet, vent needle and withdrawal port. A self-made pipet tip-to-cannula adapter allowed withdrawal of defined 25-100 µL aliquots at an activity level of ~13 MBq/µL for further processing under optimized conditions as described above. Results: The use of small reaction volumes and HPLC vials enabled an efficient 18F-optimization workflow to examine rapidly a variety of reaction parameters (>50 reactions/day) using minimal amounts of precursor. Utilizin

In the current COVID-19 pandemic that we are facing, it has become evident that the development of biosensors that can aid in the different stages of screening and surveillance: infection, disease progression and recovery, is extremely important. We developed a system that can be used for the detection of SARS-CoV-2 antibodies that are present during and after the infection. Our sensor chips were composed of six sensing devices, each of these containing interdigitated gold nanowires. The surfaces of the mentioned nanowires were functionalized with proteins of the SARS-CoV-2 so that the antibodies present in solution can bind to them. The detection of such antigen-antibody binding events was performed using the impedance spectroscopy technique. For this purposes, an AC signal over a range of frequencies (20 Hz-1 MHz) was applied to the sensor chips and the changes in impedance and phase were measured. The change in the overall impedance of the system was correlated to antigen-antibody binding events. The developed sensing system is a versatile platform that could be easily adapted to detect relevant target-analyte pairs in different diseases.

Aim/Introduction: In recent years quinoline-based small molecules targeting the fibroblast activation protein alpha (FAP) have gained interest for imaging a variety of tumor entities (1). A number of radiotracers for SPECT and PET have been developed, but so far only three FAP inhibitor (FAPI) radioligands have been reported to be radiolabeled with fluorine-18 (2,3,4). This study shows the optimization and automation of the radiofluorination of FAPI-74. Materials and Methods: (S)-(4-Carboxymethyl-7-{2-[4-(3-{4-[2-(2-cyano-pyrrolidin-1-yl)-2-oxoethylcarbamoyl]-quinolin-6-yloxy}-propyl)-piperazin-1-yl]-2-oxo-ethyl}-[1,4,7]triazonan-1-yl)-acetic acid, commonly referred to as FAPI-74, was radiolabeled using the [18F]AlF chelation method. Starting from [18F]fluoride, the reaction with AlCl3, chelate formation and subsequent purification was initially optimized by manual syntheses. Optimization included the examination of different anion exchangers (QMA light, PSHCO3) and elution solutions (NaOAc buffer pH4, 0.9% NaCl) as well as careful adjustment of the reaction parameters time (0-20 min), temperature (r.t. to 100°C), amount of AlCl3 and NaOAc buffer pH4, solvents (DMSO, EtOH), and precursor concentration (1-350 µM). Radiochemical conversion (RCC) was determined by radio-UPLC of the crude reaction mixtures. Selected reaction mixtures were analyzed after decay using UPLC-MS to identify non-radioactive byproducts. The optimized radiosynthetic procedure was transferred to a fully automated radiosynthesizer (TRACERlab FXFN) and the final product was purified and formulated using semi-preparative HPLC and SPE. Results: Under optimized conditions, RCC of [18F]AlF-FAPI-74 of > 99% was still observed at precursor concentrations as low as 12 µM FAPI-74 after reaction in a 1:1 molar ratio with AlCl3 in DMSO/sodium acetate buffer at pH 4 at 80°C for 15 minutes. Transfer of optimized conditions and upscaling was successfully achieved and delivered radiochemica

Bone defects of critical size after compound fractures, infections, or tumor resections are a challenge in treatment. Particularly, this applies to bone defects in patients with impaired bone healing due to frequently occurring metabolic diseases (above all diabetes mellitus and osteoporosis), chronic inflammation, and cancer. Adjuvant therapeutic agents such as recombinant growth factors, lipid mediators, antibiotics, antiphlogistics, and proangiogenics as well as other promising anti-resorptive and anabolic molecules contribute to improving bone healing in these disorders, especially when they are released in a targeted and controlled manner during crucial bone healing phases. In this regard, the development of smart biocompatible and biostable polymers such as implant coatings, scaffolds, or particle-based materials for drug release is crucial. Innovative chemical, physico- and biochemical approaches for controlled tailor-made degradation or the stimulus-responsive release of substances from these materials, and more, are advantageous. In this review, we discuss current developments, progress, but also pitfalls and setbacks of such approaches in supporting or controlling bone healing. The focus is on the critical evaluation of recent preclinical studies investigating different carrier systems, dual- or co-delivery systems as well as triggered- or targeted delivery systems for release of a panoply of drugs.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is closely associated with chemo-/radioresistance and poor prognosis of solid tumors. Therefore, COX-2 represents an attractive target for functional characterization of tumors by positron emission tomography (PET). In this study, the celecoxib derivative 3-([18F]fluoromethyl)-1-[4-(methylsulfonyl)phenyl]-5-(p-tolyl)-1H-pyrazole ([18F]5a) was chosen as a lead compound having a reported high COX-2 inhibitory potency and a potentially low carbonic anhydrase binding tendency. The respective deuterated analog [D2,18F]5a and the fluoroethyl-substituted derivative [18F]5b were selected to study the influence of these modifications with respect to COX inhibition potency in vitro and metabolic stability of the radiolabeled tracers in vivo. COX-2 inhibitory potency was found to be influenced by elongation of the side chain but, as expected, not by deuteration. An automated radiosynthesis comprising 18F-fluorination and purification under comparable conditions provided the radiotracers [18F]5a,b and [D2,18F]5a in good radiochemical yields (RCY) and high radiochemical purity (RCP). Biodistribution and PET studies comparing all three compounds revealed bone accumulation of 18F-activity to be lowest for the ethyl derivative [18F]5b. However, the deuterated analog [D2,18F]5a turned out to be the most stable compound of the three derivatives studied here. Time-dependent degradation of [18F]5a,b and [D2,18F]5a after incubation in murine liver microsomes was in accordance with the data on metabolism in vivo. Furthermore, metabolites were identified based on UPLC-MS/MS.

Due to the involvement of cyclooxygenase-2 (COX-2) in carcinogenesis, COX-2-selective inhibitors are increasingly studied for their potential cytotoxic properties. Moreover, the incorporation of carboranes in structures of established anti-inflammatory drugs can improve the potency and metabolic stability of the inhibitors. Herein, we report the synthesis of carborane-containing derivatives of rofecoxib that display remarkable cytostatic activity in the micromolar range with excellent selectivity for melanoma and colon cancer cell lines over normal cells. Furthermore, it was shown that the carborane-modified derivatives of rofecoxib displayed different modes of action that was dependent on the cell type.

Non-invasive imaging of cyclooxygenase-2 (COX-2) by radiolabeled ligands is attractive for the diagnosis of cancer and novel highly affine leads with optimized pharmacokinetic profile are of high interest for future developments. Recent findings have shown that methylsulfonyl-substituted (dihydro)pyrrolo[3,2,1-hi]indoles represent highly potent and selective COX-2 inhibitors but possess unsuitable pharmacokinetic properties for radiotracer applications. Based on these results, we herein present the development and evaluation of a second series of sulfonamide-substituted (dihydro)pyrrolo[3,2,1-hi]indoles and their conversion into the respective more hydrophilic N-propionamide-substituted analogs. In comparison to the methylsulfonyl-substituted leads, COX inhibition potency and selectivity was retained in the sulfonamide-substituted compounds; however, the high lipophilicity might hinder their future use. The N-propionamide-substituted analogs showed a significantly decreased lipophilicity and, as expected, lower or no COX-inhibition potency. Hence, the N-(sulfonyl)propionamides can be regarded as potential prodrugs, which represents a potential approach for more sophisticated radiotracer developments.

Ketoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) that also exhibits cytotoxic activity against various cancers. This makes ketoprofen an attractive structural lead for the development of new NSAIDs and cytotoxic agents. Recently, the incorporation of carboranes as phenyl mimetics in structures of established drugs has emerged as an attractive strategy in drug design. Herein, we report the synthesis and evaluation of four novel carborane-containing derivatives of ketoprofen, two of which are prodrug esters with an nitric oxide-releasing moiety. One of these prodrug esters exhibited high cytostatic activity against melanoma and colon cancer cell lines. The most pronounced activity was found in cell lines that are sensitive to oxidative stress, which was apparently induced by the ketoprofen analogue.