Ferrocenyl-coupled n-heterocyclic carbene complexes of gold(i): a successful approach to multinuclear anticancer drugs

Four gold(I) carbene complexes featuring 4-ferrocenyl substituted imidazol-2-ylidene ligands were investigated for antiproliferative and antivascular properties. They were active against a panel of seven cancer cell lines, including multidrug-resistant ones, with low micromolar or nanomolar IC50 (72 h) values, according to their lipophilicity and cellular uptake. The delocalised lipophilic cationic complexes 8 and 10 acted by increasing the reactive oxygen species in two ways: via a genuine ferrocene effect and by inhibiting the thioredoxin reductase. Both complexes gave rise to a reorganization of the F-actin cytoskeleton in endothelial and melanoma cells, associated with a G1 phase cell cycle arrest and a retarded cell migration. They proved antiangiogenic in tube formation assays with endothelial cells and vascular-disruptive on real blood vessels in the chorioallantoic membrane of chicken eggs. Biscarbene complex 10 was also tolerated well by mice where it led to a volume reduction of xenograft tumors by up to 80%

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvβ3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvβ3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvβ3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application

Squaric Ester-Based, pH-Degradable Nanogels:Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.</p

Hyperprolactinemia in adults with Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a rare neurodevelopmental genetic disorder typically characterized by body composition abnormalities, hyperphagia, behavioural challenges, cognitive dysfunction, and hypogonadism. Psychotic illness is common, particularly in patients with maternal uniparental disomy (mUPD), and antipsychotic medications can result in hyperprolactinemia. Information about hyperprolactinemia and its potential clinical consequences in PWS is sparse. Here, we present data from an international, observational study of 45 adults with PWS and hyperprolactinemia. Estimated prevalence of hyperprolactinemia in a subset of centres with available data was 22%, with 66% of those related to medication and 55% due to antipsychotics. Thirty-three patients were men, 12 women. Median age was 29 years, median BMI 29.8 kg/m2, 13 had mUPD. Median prolactin was 680 mIU/L (range 329–5702). Prolactin levels were higher in women and patients with mUPD, with only 3 patients having severe hyperprolactinemia. Thyroid function tests were normal, 24 were treated with growth hormone, 29 with sex steroids, and 20 with antipsychotic medications. One patient had kidney insufficiency, and one a microprolactinoma. In conclusion, severe hyperprolactinemia was rare, and the most common aetiology of hyperprolactinemia was treatment with antipsychotic medications. Although significant clinical consequences could not be determined, potential negative long-term effects of moderate or severe hyperprolactinemia cannot be excluded. Our results suggest including measurements of prolactin in the follow-up of adults with PWS, especially in those on treatment with antipsychotic

In vivo gene silencing in the liver with siRNA loaded non-biodegradable and biodegradable cationic nanohydrogel particles for antifibrotic therapy

Cationic nanohydrogel particles are interesting vehicles for systemic siRNA delivery. For the first time, optimized anti-procollagen α1(I) siRNA loaded cationic nanohydrogel particles were tested in vivo as an antifibrotic therapy in a mouse model of liver fibrosis, induced by oral gavage of CCl4. After thorough validation of siRNA loaded nanohydrogel particles in vitro for cell toxicity, cellular uptake and knockdown performance, particles qualified for further biological evaluation in vivo. After systemic administration, siRNA loaded particles almost exclusively accumulated in liver, while lungs, spleen and kidneys were only targeted to a minor extend. On the cellular level, complexes addressed primarily activated myofibroblasts, the major collagen producing cells in liver fibrosis, and to a lesser extend endothelial cells and macrophages. Anti-procollagen α1(I) siRNA loaded cationic nanohydrogel sequence specifically and significantly suppressed hepatic procollagen α1(I) transcript levels and total collagen accumulation in liver fibrotic mice. Furthermore, a significant reduction of α-SMA, a surrogate marker of myofibroblast activation, supported the potent antifibrotic effect of the anti-procollagen α1(I) siRNA/nanoparticle treatment. Thus, cationic nanohydrogel particles qualified as promising vehicle platform for siRNA delivery to nonparenchymal liver cells. Since these first generation cationic nanohydrogel particles proved to be an attractive tool for systemic siRNA delivery, we attempted to improve their in vivo degradability and thus tolerability. This improvement is desirable, especially to prevent potential long-term side effects by tissue and cellular accumulation. A second generation of novel nanohydrogel particles was generated that contained a cationic biodegradable acid-labile cross-linker, endowing the next generation of nanohydrogel particles with enhanced biodegradability (bioNP). The first generation (non-bioNP) were compared 1:1 vs. the second generation cationic nanohydrogel particles (bioNP), loaded with anti-procollagen α1(I) siRNA, in another set of liver fibrosis studies. In vitro, acid-labile biodegradable complexes loaded with anti-procollagen α1(I) siRNA could compete with non-biodegradable complexes and showed no cytotoxicity (up to 400 nM siRNA), high cellular uptake (~100% after 24h) and an even improved knockdown efficiency for procollagen 1α(I) transcript mRNA (up to 80% for bioNP or 60% for non-bioNP) in murine fibroblasts. In vivo biodistribution in CCl4 induced liver fibrotic mice, using near infrared (NIR) imaging of NP that were labeled with two different NIR-dyes (one coupled to the siRNA, one coupled to the NP shell) showed an equally efficient siRNA delivery to liver and liver resident myofibroblasts for both NP species. However, in vivo half-lives of NP and their siRNA cargo differed between the first and second generation of cationic nanohydrogel particles. After repetitive iv injection BioNP/siRNA-complexes exhibited, compared to their first generation, an enhanced biodegradability with less tendency to (over)-accumulate in fibrotic and healthy livers. Nevertheless, anti-procollagen α1(I) siRNA loaded bioNP induced a potent procollagen α1(I) mRNA knockdown and prevented fibrosis progression as determined by liver biochemical and morphometrical collagen quantification. Long-term monitoring of the carriers in the body revealed a significantly enhanced clearance for the acid-degradable carrier, especially after multiple dosing. In healthy mice, both species showed a broad therapeutic index in doses escalation studies up to 10mg siRNA/kg body weight. Therefore, the novel acid-degradable cationic bio-NP could be validated as a promising novel platform for siRNA delivery in vivo to treat fibrotic liver diseases. Since these NP also target other nonparenchymal cells, especially macrophages and endothelial cells, further studies to deliver siRNA to these cells are warranted.Kationische Nanopartikel (NP) sind interessante Vehikel zur systemischen Applikation von siRNA. Wir testeten in vivo zum ersten Mal optimierte anti-procollagen α1(I) siRNA beladene NP zur antifibrotischen Therapie in CCl4 induzierten leberfibrotischen Mäusen. Nach gründlicher in vitro Untersuchung der anti-procollagen 1α(I) siRNA beladenen Partikel hinsichtlich ihrer Zytotoxizität, Zellaufnahme und ihrer Knockdown-Effizienz (in 3T3-Fibroblasten), konnten sie sich als vielversprechende siRNA Träger für in vivo Versuche qualifizieren. Nach intravenöser Applikation akkumulierten die siRNA beladenen Partikel beinahe ausschließlich in der Leber und nur marginal in Lunge, Milz und Niere. Auf zellulärer Ebene wurden die Komplexe primär von aktivierten Myofibroblasten und nur zu einem kleinen Teil von Endothelzellen, Makrophagen und Hepatozyten aufgenommen. Anti-procollagen α1(I) siRNA beladene NP reduzierten signifikant und sequenzspezifisch hepatisches prokollagen α1(I) mRNA Transkript, sowie die Gesamtmenge an Kollagen in leberfibrotischen Mäusen. Des Weiteren zeigte die Reduktion von α-SMA, ein Surrogatemarker für aktivierte Myofibroblasten, einen Erfolg der antifibrotischen Therapie. Somit qualifizierten sich NPs als geeignete siRNA Träger zur Adressierung nicht-parenchymale Zellen in der Leber. Da die erste Generation von NP/anti-procollagen α1(I) siRNA Komplexen bereits überzeugende Therapierfolge in fibrotischen Mäusen zeigte, versuchten wir ihre Bioabbaubarkeit und somit ihre Verträglichkeit zu verbessern. Eine verbesserte Abbaubarkeit der Partikel war wünschenswert, um bei längeren Anwendungen toxische Akkumulationen in Gewebe und Zellen zu verhindern. Für die zweite Generation von NP wurde ein säurelabiler Ketal-Linker, anstelle des vorherigen inerten Spermin-Linkers, zur Quervernetzung verwendet. Im Rahmen einer in vivo Vergleichsstudie wurde die erste Generation (nicht-bioNP) vs der zweiten Generation (bioNP), als Träger für anti-procollagen 1α(I) siRNA, verglichen. In vitro zeigten anti-procollagen 1α(I) siRNA beladene bioNP gegenüber nicht-bioNP keine Nachteile hinsichtlich Zytotoxizität (bis zu 400nM siRNA) und Zellaufnahme (~100% nach 24h). BioNP/anti-procollagen 1α(I)1 Komplexe wiesen bemerkenswerterweise einen effizienteren Knockdown für procollagen 1α(I) mRNA Transkript (bis zu 80% für bioNP) in 3T3 Fibroblasten auf. Beide Partikelspezies zeigten, nach intravenöser Injektion in (leber-)fibrotischen Mäusen, hinsichtlich ihrer in vivo Biodistribution (IVIS-imaging) für den carrier (NP) und ihr siRNA cargo (NP und siRNA mit verschiedenen nahinfraroten Fluorochromen markiert) eine vergleichbare effiziente Anreicherung in der Leber, als auch auf zellulärer Ebene in aktivierte Myofibroblasten (α-SMA+ Zellen). Die in vivo Halbwertszeit der säurelabilen bioNP/siRNA-Komplexe konnte, im Vergleich zu ihren Vorgängern, signifikant reduziert werden. Nach Akkumulation in der Leber wurden die bioNP/siRNA-Komplexe deutlich schneller abgebaut. Die verbesserte Abbaubarkeit der bioNP ging nicht auf Kosten ihrer therapeutischen Knockdown-Effizienz. Anti-procollagen 1α(I) siRNA beladene bioNP versus non-bioNP/ anti-procollagen 1α(I) siRNA Komplexe erzielten eine vergleichbare effiziente Reduktion des procollagen 1α(I) mRNA Transkripts als auch der Gesamtkollagenlast in fibrotischen Lebern. Im Rahmen eines Langzeit-in vivo-Monitorings (über 13 Tage) wiesen die säurelabilen siRNA Träger der zweiten Generation, nach wiederholter intravenöser Applikation, eine signifikant verbesserte Bioabbaubarkeit im Vergleich zu ihren Vorgängern auf. Beide siRNA beladene Partikelspezies wurden bei Dosis-Eskalationen bis zu 10mg/kg siRNA Körpergewicht von gesunden Mäusen sehr gut vertragen und lassen daher eine große therapeutische Breite vermuten. Die NP der zweiten Generation behielten alle positiven Eigenschaften ihrer Vorgänger bei und zeigten zusätzlich eine deutlich verbesserte in vivo Bioabbaubarkeit. Somit stellen bioNP, als eine vielversprechende Weiterentwicklung ihrer ersten Generation, eine neue Plattform für siRNA Träger dar. Da die NP, neben aktivierten Myofibroblasten, zusätzlich andere nicht-parenchymale Zellen insbesondere Makrophagen und Endothelzellen erreichen, können weitere therapeutische Ansätze mit ihnen als in vivo siRNA Träger ausgelotet werden

Great Expectations: Principal Investigator and Trainee Perspectives on Hiring, Supervision, and Mentoring

A functioning mentor–trainee relationship is of high importance in academia. Discrepancies in expectations between principal investigators (PIs) and trainees are a source of misunderstandings and conflicts, endangering scientific progress and career advancement. In this pilot study, we sought to explore the expectations of PIs and trainees, providing consensus data from physician‐scientists and junior researchers who attended an educational workshop, entitled “The EASL/AASLD Masterclass,” in December 2017. Twenty‐three Masterclass attendees, comprising nine trainees (four Ph.D. candidates, five postdoctoral researchers) and 14 PIs, responded to an online survey. Both parties were asked to score 29 predefined statements of important expectations, enabling a comparative analysis for each statement between the groups. For the trainees, the success of the PI, either mirrored by successful mentoring or scientific work, as well as a clear road for academic development are of utmost importance. PIs did not prioritize these aspects, highlighting discrepancies of expectations. PIs prioritized trainee competence, reliability, and strong daily initiative/work ethic, qualities that were also recognized to be important by the trainee group but not to same degree as PIs. Conclusion:Discrepancies in expectations pose a preventable threat to the mentor–trainee relationship if considered and discussed beforehand. The discrepancy in the most common expectations between the two groups could have resulted from the fact that trainees prioritize outcomes of success while PIs focus on the necessary qualities leading to those outcomes

Reductive decationizable block copolymers for stimuli-responsive mRNA delivery

Messenger ribonucleic acids (mRNAs) are considered as promising alternatives for transient gene therapy, but to overcome their poor pharmacokinetic properties, smart carriers are required for cellular uptake and stimuli-responsive release. In this work, a synthetic concept toward reductive decationizable cationic block copolymers for mRNA complexation is introduced. By combination of RAFT block copolymerization with postpolymerization modification, cationic block copolymers are generated with disulfide-linked primary amines. They allow effective polyplex formation with negatively charged mRNA and subsequent release under reductive conditions of the cytoplasm. In first in vitro experiments with fibroblasts and macrophages, tailor-made block copolymers mediate cellspecific mRNA transfection, as quantified by polyplex uptake and mRNA-encoding gene expression. Furthermore, RAFT polymerization provides access to heterotelechelic polymers with orthogonally addressable endgroup functionalities utilized to ligate targeting units onto the polyplex-forming block copolymers. The results exemplify the broad versatility of this reductive decationizable mRNA carrier system, especially toward further advanced mRNA delivery applications

The Role of p53 Dysfunction in Colorectal Cancer and Its Implication for Therapy

Colorectal cancer (CRC) is one of the most common and fatal cancers worldwide. The carcinogenesis of CRC is based on a stepwise accumulation of mutations, leading either to an activation of oncogenes or a deactivation of suppressor genes. The loss of genetic stability triggers activation of proto-oncogenes (e.g., KRAS) and inactivation of tumor suppression genes, namely TP53 and APC, which together drive the transition from adenoma to adenocarcinoma. On the one hand, p53 mutations confer resistance to classical chemotherapy but, on the other hand, they open the door for immunotherapy, as p53-mutated tumors are rich in neoantigens. Aberrant function of the TP53 gene product, p53, also affects stromal and non-stromal cells in the tumor microenvironment. Cancer-associated fibroblasts together with other immunosuppressive cells become valuable assets for the tumor by p53-mediated tumor signaling. In this review, we address the manifold implications of p53 mutations in CRC regarding therapy, treatment response and personalized medicine