Highly Fluorescent Distyrylnaphthalene Derivatives as a Tool for Visualization of Cellular Membranes

Fluorescent imaging, which is an important interdisciplinary field bridging research from organic chemistry, biochemistry and cell biology has been applied for multi-dimensional detection, visualization and characterization of biological structures and processes. Especially valuable is the possibility to monitor cellular processes in real time using fluorescent probes. In this work, conjugated oligoelectrolytes and neutral derivatives with the distyrylnaphthalene core (SN-COEs) were designed, synthetized and tested for biological properties as membrane-specific fluorescent dyes for the visualization of membrane-dependent cellular processes. The group of tested compounds includes newly synthesized distyrylnaphthalene derivatives (DSNNs): a trimethylammonium derivative (DSNN-NMe3+), a phosphonate derivative (DSNN-P), a morpholine derivative (DSNN-Mor), a dihydroxyethylamine derivative (DSNN-DEA), a phosphonate potassium salt (DSNN-POK), an amino derivative (DSNN-NH2) and pyridinium derivative (DSNN-Py+). All compounds were tested for their biological properties, including cytotoxicity and staining efficiency towards mammalian cells. The fluorescence intensity of SN-COEs incorporated into cellular structures was analyzed by fluorescence activated cell sorting (FACS) and photoluminescence spectroscopy. The cytotoxicity results have shown that all tested SN-COEs can be safely used in the human and animal cell studies. Fluorescence and confocal microscopy observations confirm that tested COEs can be applied as fluorescent probes for the visualization of intracellular membrane components in a wide range of different cell types, including adherent and suspension cells. The staining procedure may be performed under both serum free and complete medium conditions. The presented studies have revealed the interesting biological properties of SN-COEs and confirmed their applicability as dyes for staining the membranous structures of eukaryotic cells, which may be useful for visualization of wide range of biological processes dependent of the extra-/intracellular communications and/or based on the remodeling of cellular membranes

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3′-Iron-1,2,1′,2′-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression

EGFR-Targeted Cellular Delivery of Therapeutic Nucleic Acids Mediated by Boron Clusters

New boron carriers with high boron content and targeted cancer-cell delivery are considered the first choice for boron neutron capture therapy (BNCT) for cancer treatment. Previously, we have shown that composites of antisense oligonucleotide and boron clusters are functional nanoparticles for the downregulation of expression of epidermal growth factor receptor (EGFR) and can be loaded into EGFR-overexpressing cancer cells without a transfection factor. In this study, we hypothesize that free cellular uptake is mediated by binding and activation of the EGFR by boron clusters. Proteomic analysis of proteins pulled-down from various EGFR-overexpressing cancer cells using short oligonucleotide probes, conjugated to 1,2-dicarba-closo-dodecaborane (1,2-DCDDB, [C2B10H12]) and [(3,3′-Iron-1,2,1′,2′-dicarbollide)−] (FESAN, [Fe(C2B9H11)2]−), evidenced that boron cage binds to EGFR subdomains. Moreover, inductively coupled plasma mass spectrometry (ICP MS) and fluorescence microscopy analyses confirmed that FESANs-highly decorated B-ASOs were efficiently delivered and internalized by EGFR-overexpressing cells. Antisense reduction of EGFR in A431 and U87-MG cells resulted in decreased boron accumulation compared to control cells, indicating that cellular uptake of B-ASOs is related to EGFR-dependent internalization. The data obtained suggest that EGFR-mediated cellular uptake of B-ASO represents a novel strategy for cellular delivery of therapeutic nucleic acids (and possibly other medicines) conjugated to boron clusters