Leonard Kaps

Gold(I) Biscarbene Complexes Derived from Vascular‐Disrupting Combretastatin A‐4 Address Different Targets and Show Antimetastatic Potential

Gold N‐heterocyclic carbene (NHC) complexes are an emerging class of anticancer drugs. We present a series of gold(I) biscarbene complexes with NHC ligands derived from the plant metabolite combretastatin A‐4 (CA‐4) that retain its vascular‐disrupting effect, yet address different cellular and protein targets. Unlike CA‐4, these complexes did not interfere with tubulin, but with the actin cytoskeleton of endothelial and cancer cells. For the highly metastatic 518A2 melanoma cell line this effect was accompanied by a marked accumulation of cells in the G1 phase of the cell cycle and a suppression of active prometastatic matrix metalloproteinase‐2. Despite these mechanistic differences the complexes were as strongly antivascular as CA‐4 both in vitro in tube formation assays with human umbilical vein endothelial cells, and in vivo as to blood vessel disruption in the chorioallantoic membrane of chicken eggs. The antiproliferative effect of the new gold biscarbene complexes in a panel of six human cancer cell lines was impressive, with low sub‐micromolar IC50 values (72 h) even against CA‐4‐refractory HT‐29 colon and multidrug‐resistant MCF‐7 breast carcinoma cells. In preliminary studies with a mouse melanoma xenograft model the complexes led to significant decreases in tumor volume while being very well tolerated.

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 delocalized lipophilic cationic complexes 8 and 10 acted by increasing the reactive oxygen species in two ways: through 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 %.

In vivo gene-silencing in fibrotic liver by siRNA-loaded cationic nanohydrogel particles

Cationic nanohydrogel particles loaded with anti-Col1α1 siRNA suppress collagen synthesis and deposition in fibrotic mice: Systemically administered 40 nm sized nanogel particles accumulate in collagen-expressing cells in the liver. Their siRNA payload induces a sequence specific in vivo gene knockdown affording an efficient antifibrotic effect in mice with liver fibrosis.

SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles

Abstract Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross‐linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo‐amine cross‐linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross‐linking of self‐assembled reactive ester block copolymers in DMSO. The resulting acid‐sensitive cationic nanoparticles spontaneously disassembled over time in acidic milieu, as investigated by dynamic light scattering. Fluorescent correlation spectroscopy showed effective complexation with siRNA as well as its release upon particle degradation at endosomal pH. These properties resulted in an enhanced in vitro gene knockdown for the acid‐degradable cationic nanoparticles compared to their non‐degradable spermine analogues. In a murine liver fibrosis model enhanced carrier and payload accumulation in the fibrotic tissue facilitated sequence‐specific gene knockdown and prevented fibrosis progression. Long‐term monitoring of the carrier in the body showed an enhanced clearance for the acid‐degradable carrier, even after multiple dosing. Therefore, these acid‐degradable cationic nanohydrogel particles can be considered as promising siRNA carriers for in vivo purposes towards therapeutic applications. Graphical abstract Figure. No Caption available.

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 cell-specific 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.

Synergistic "gold effects" of anti-vascular 4,5-diarylimidazol-2-ylidene gold(I) carbene complexes.

The natural cis-stilbene combretastatin A-4 (CA-4) is a vascular disrupting agent (VDA) that leads to a vascular shutdown in solid tumors. Its activity originates from an inhibition of the polymerization of tubulin and of the functionality of the VE-cadherin/ β-catenin complex which is crucial for cell– cell adhesion [1]. However, the insufficient cytotoxicity of CA-4 and its chemical instability in solution limits its clinical usability. A more water-soluble phosphate prodrug of CA-4 is currently undergoing clinical trials [2]. Derivatives of CA-4 with improved chemical stability and retained anti-vascular effect were obtained by incorporation of the Z-alkene in heterocycles such as imidazoles [3]. Since structurally simple imidazolium salts had already been employed as ligand precursors for anticancer N-heterocyclic carbene (NHC) complexes [4], we prepared gold carbene complexes that combine the intrinsic anti-vascular activity of CA-4 analogous imidazoles 1 (Figure 1A) with the known anticancer properties of gold fragments [5]. Previously, we reported on the cytotoxicity and the cellular uptake of such new gold(I)-NHC complexes [5]. Now we present first insights into the mechanism of action of two such complexes, 2a and 3a (Figure 1A).

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

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.

Enhanced protection of C57 BL/6 vs Balb/c mice to melanoma liver metastasis is mediated by NK cells

ABSTRACT The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediated cytotoxicity against B16F10 cells. Our findings highlight the role of NK cells in innate anti-tumor immunity in the context of the liver – particularly against highly aggressive MHC I-deficient cancer cells. Moreover, the B16F10 model of melanoma liver metastasis is suited for developing novel therapies targeting innate NK cell related immunity in liver metastases and liver cancer.

P0312 : Preclinical evaluation of dextran-based therapeutic nanoparticles for hepatic drug delivery

AIM Evaluation of dextran-based nanoparticles (DNP) as a drug delivery system to target myeloid cells of the liver. MATERIALS & METHODS DNP were synthesized and optionally PEGylated. Their toxicity and cellular uptake were studied in vitro. Empty and siRNA-carrying DNP were tested in vivo with regard to biodistribution and cellular uptake. RESULTS In vitro, DNP were taken up by cells of the myeloid lineage without compromising their viability. In vivo, empty and siRNA-carrying DNP distributed to the liver where a single treatment addressed approximately 70% of macrophages and dendritic cells. Serum parameters indicated no in vivo toxicity. CONCLUSION DNP are multifunctional liver-specific drug carriers which lack toxic side effects and may be utilized in clinical applications targeting liver macrophages.