In Vitro Evaluation of Non-Protein Adsorbing Breast Cancer Theranostics Based on 19F-Polymer Containing Nanoparticles

Eight fluorinated nanoparticles (NPs) are synthesized, loaded with doxorubicin (DOX), and evaluated as theranostic delivery platforms to breast cancer cells. The multifunctional NPs are formed by self-assembly of either linear or star-shaped amphiphilic block copolymers, with fluorinated segments incorporated in the hydrophilic corona of the carrier. The sizes of the NPs confirm that small circular NPs are formed. The release kinetics data of the particles reveals clear hydrophobic core dependence, with longer sustained release from particles with larger hydrophobic cores, suggesting that the DOX release from these carriers can be tailored. Viability assays and flow cytometry evaluation of the ratios of apoptosis/necrosis indicate that the materials are non-toxic to breast cancer cells before DOX loading; however, they are very efficient, similar to free DOX, at killing cancer cells after drug encapsulation. Both flow cytometry and confocal microscopy confirm the cellular uptake of NPs and DOX-NPs into breast cancer cells, and in vitro 19F-MRI measurement shows that the fluorinated NPs have strong imaging signals, qualifying them as a potential in vivo contrast agent for 19F-MRI

Enzymatic production of monoclonal stoichiometric single stranded DNA oligonucleotides.

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides

DNA interaction of CuII, NiII and ZnII functionalized salphen complexes: studies by linear dichroism, gel electrophoresis and PCR

The interaction of salphen-type NiII, CuII and ZnII complexes with native DNA was investigated by exploiting linear dichroism experiments. The NiII complex behaves as a typical intercalator, binding strongly and stiffening and unwinding the DNA. The strength of the DNA interaction is slightly weaker for the copper complex and much weaker for the zinc complex. Plasmid-DNA gel electrophoresis experiments indicated that while CuII and ZnII complexes do not induce the unwinding of supercoiled DNA, the NiII complex has a nuclease activity without the addition of external agents. On the other hand, as shown in the PCR assays, we demonstrate that, at the used concentrations, only the CuII complex is able to inhibit the DNA amplification mediated by Taq DNA polymerase. In this paper we have also reported a detailed characterization of the three compounds including 2D-NMR and ESI-mass experiments and X-ray single crystal structure of the copper and nickel compounds

Interplay of three G‑quadruplex units in the KIT promoter

The proto-oncogene KIT encodes for a tyrosine kinase receptor, which is a clinically validated target for treating gastrointestinal stromal tumors. The KIT promoter contains a G-rich domain within a relatively long sequence potentially able to form three adjacent G-quadruplex (G4) units, namely, K2, SP, and K1. These G4 domains have been studied mainly as single quadruplex units derived from short truncated sequences and are currently considered promising targets for anticancer drugs, alternatively to the encoded protein. Nevertheless, the information reported so far does not contemplate the interplay between those neighboring G4s in the context of the whole promoter, possibly thwarting drug-discovery efforts. Here we report the structural and functional study of the KIT promoter core sequence, in both single- and double-stranded forms, which includes all three predicted G4 units. By preventing the formation of alternatively one or two G4 units and by combining biophysical techniques and biological assays, we show for the first time that these quadruplexes cannot be analyzed independently, but they are correlated to each other. Our data suggest that, while K2 and K1 G-rich sequences retain the ability to fold into parallel G4 motifs within a long sequence, the SP G-rich domain contributes to G4 structure only together with K2. Remarkably, we have found that, in the context of a dynamic equilibrium between the three G4 units, the G4 formed by K1 has the most significant influence on the structure stability and on the biological role of the whole promoter

DNA Binding Studies and Cytotoxicity of a Dinuclear PtII Diazapyrenium- Based Metallo-supramolecular Rectangular Box

The interaction with native DNA of a 2,7-diazapyrenium-based ligand 1 and its PtII rectangular metallacycle 2 is explored through circular and linear dichroism and fluorescence spectroscopies. The metal-free ligand 1 binds through intercalation, with a binding constant of approximately 5*105m-1, whereas the metallacycle 2 binds and bends the DNA with a binding constant of 7*106m-1. PCR assays show that metallo-supramolecular box 2 interferes with DNA transactions in vitro whereas the intercalator 1 does not. The metallacycle is active against four human cancer cell lines, with IC50 values ranging between 3.1 and 19.2 mm and shows similar levels of efficacy, but a different spectrum of activity, to cisplatin

Predicting the Three-Dimensional Structure of the c-KIT Proto-Oncogene Promoter and the Dynamics of Its Strongly Coupled Guanine Quadruplexes

Guanine quadruplexes (G4s) play essential protective and regulatory roles within cells, influencing gene expression. In several gene-promoter regions, multiple G4-forming sequences are in close proximity and may form three-dimensional arrangements. We analyze the interplay among the three neighboring G4s in the c-KIT proto-oncogene promoter (WK1, WSP, and WK2). We highlight that the three G4s are structurally linked and their cross-talk favors the formation of a parallel structure for WSP. Relying on all-atom molecular dynamic simulations exceeding the μs time scale and using enhanced sampling methods, we provide the first computationally resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that can ultimately be used as a starting point for drug discovery

Selective G-Quadruplex Stabilizers: Schiff-base Metal Complexes with Anticancer Activity

The affinity of three square-planar nickel(II) (1), copper(II) (2) and zinc(II) (3) Schiff-base complexes for wildtype human telomeric (h-Telo) and protooncogene c-myc G-quadruplex (G4) DNA was investigated by UVvisible absorption spectroscopy and circular dichroism. DNA-binding constants (Kb) were determined by spectrophotometric titrations for both G4-DNA and B-DNA. The results obtained point out that the three metal complexes selectively bind G4-DNA with higher affinity, up to two orders of magnitude, with respect to B-DNA. The nickel(II) complex 1 was found to be the most effective G4-DNA stabilizer and the Kb values decrease in the order 1 > 2 z 3. Innovative computational investigations, consisting of molecular dynamics (MD) simulations followed by density functional theory/molecular mechanics (DFT/MM) calculations, provide atomistic support for the interpretation of the binding mechanism to G4-DNA by end stacking and also of the experimental affinity order. Interestingly, 1 is able to induce G4-DNA formation of h-Telo sequences, also in the absence of K+ cations. This last result is nicely confirmed and highlighted by polymerase chain reaction (PCR) stop assays, which show the ability of the title compounds to induce and stabilize G4 structures inhibiting the amplification of PCR products. Finally, compounds 1–3 showed concentration and time-dependent cytotoxicity towards HeLa and MCF-7 human cancer cell lines, inducing significant effects on cell cycle distribution with G2/M arrest in HeLa cells and G0/G1 arrest in MCF-7 cells. Overall, the PCR inhibition and anticancer activity of the three compounds decreases in the same order 1 > 2 z 3, in excellent correlation with the G4-DNAbinding affinity, implying that G4-DNA is the biotarget for their biological activity