Combination treatments in <i>in vitro</i> and <i>in vivo</i> models between molecules reverting epigenetic gene silencing and DNA-interacting anticancer drugs

Epigenetic transcriptional gene silencing plays a fundamental role in cancer development and has been considered as a target for cancer therapy in the last few years, mainly due to its reversibility by small molecules. Among the several methylated genes investigated, glutathione-S-transferase (GST) P1, a protein belonging to cellular detoxification systems, has been shown to be extensively promoter-methylated in prostate cancer. My study therefore describes a new therapeutic approach against prostate cancer, based on the combination of demethylating agents and brostallicin, a DNA minor groove binding drug, which is activated in the cell by binding to glutathione, a reaction catalyzed by GST. Among the demethylating molecules tested on the prostatic cancer cell line LNCaP in in vitro combinations with brostallicin, zebularine was able to increase brostallicin activity with little toxicity compared to the other tested demethylating drugs. These in vitro results prompted the in vivo testing of zebularine with brostallicin on LNCaP cells transplanted in mice. Prolonged treatment with zebularine was able to significantly improve brostallicin antitumour activity compared to both drugs administrated as single agents. When GSTP1 expression was investigated in treated samples versus untreated controls, no protein re-expression was found and this was related to the unchanged levels of GSTP1 promoter methylation. In contrast, the demethylating effect of zebularine was clearly evident in the promoter of GSTM1 gene, which is also silenced by methylation in LNCaP cells. GSTM1 codes for a class of GST enzymes that has recently been found to be more active on brostallicin than GSTP1. This indicates that the activation of brostallicin cytotoxicity in LNCaP cells by zebularine likely depends on enzymatic activation by GSTM1 rather than GSTP1 and strengthens the feasibility of this combination as a treatment for prostate cancer in the clinic, and as model for the therapy of other solid tumours

Inherently fluorescent polyaniline nanoparticles in a dynamic landscape

In this paper we report for the first time on the emissive behavior of two polyaniline (PANI) nanoparticle systems produced via oxidative chemical polymerization in the presence of either poly(vinyl alcohol)(PVA) or chitosan as polymeric stabilizers in water. The emission from PANI nanoparticles is irreversibly quenched by an increase of pH of the suspending medium from acid to neutral (chitosan–PANI) or alkaline (PVA–PANI). Conversely, PANI nanorods synthesized in the same conditions of the above, but in presence of poly(N-vinyl pyrrolidone), is not emissive at any pH. The role of the polymeric surfactant as a soft template is key in controlling the morphology and the properties of the obtained PANI dispersions. FTIR, UV–Vis absorption and photoluminescence excitation (PLE) spectra studies suggest that the emissive properties are related to the establishment of strong, non-covalent interactions between nanoscalar PANI particles and the polymeric surfactant at the pH of synthesis. Morphology examination of the three systems, by both dynamic light scattering (DLS) and Transmission Electron Microscopy (TEM), reveal that photoluminescence is associated to the presence of a genuinely 3D nanoscalar morphology, together with an ordered disposition of PANI chains into aligned crystal planes. Concomitant to the irreversible quenching of the emission signal with increasing pH, there is an evolution of the morphology leading to particle coalescence, coarsening and ultimately phase-separation, with consequent modification of PANI–polymeric surfactant interactions, PANI chains supra-molecular organization and optical properties of the PANI nanoparticles dispersion

Glutathione-sensitive nanogels for drug release

Nanogels (NGs) synthesized by pulsed electron-beam irradiation of semi-dilute poly (N-vinyl pyrrolidone) (PVP) aqueous solutions, at relatively low energy per pulse and doses within the sterilization dose range, represent a very interesting family of polymeric nanocarriers. Ionizing irradiation-induced crosslinking of PVP allows to control particle size, and surface chemistry of the polymer nanoparticles without making use of catalysts, organic solvents or surfactants, and with beneficial effects onto the purity and hence biocompatibility of the final products obtained. Furthermore, the availability of reactive functional groups, either generated by the radiation or purposely grafted via copolymerisation with suitable functional monomers enables the conjugation of therapeutics drug, that make them suitable nanocarriers for biomedical applications. In particular, we have developed a carboxyl-functionalized nanogel variant for glutathione-mediated delivery of a chemotherapeutic agent, Doxorubicin. The drug is linked to the nanoparticles through a linker containing a cleavable disulphide bridge, aminoethyldithiopropionic acid (AEDP). In vitro drug release experiments have shown that glutathione can induce the release of Doxorubicin, through the reduction of the disulfide bridge. These results suggest that such redox-responsive nanoparticles can deliver doxorubicin into the nuclei of tumor cells, thus inducing inhibition of cell proliferation, and provide a favourable platform to construct nanoscalar drug delivery systems for cancer therapy

Down-regulation of the Nucleotide Excision Repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells

<p>Abstract</p> <p>Background</p> <p>Drug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy.</p> <p>Results</p> <p>We present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter.</p> <p>Conclusions</p> <p>Methylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.</p

Synthesis of polymer nanogels by electro-Fenton process: investigation of the effect of main operation parameters

Recently, electro-Fenton (EF) process has been shown as a promising, facile, effective, low cost and environmentally-friendly alternative for synthesizing polymer nanogels suitable as biocompatible nanocarriers for emerging biomedical applications. Here, the electrochemically-assisted modification of poly(vinylpyrrolidone) (PVP) by EF process was studied to assess the role of key operation parameters for a precise modulation of polymer crosslinking and its functionalization with -COOH and succinimide groups. The dimensions of the nanogels, in terms of hydrodynamic radius (Rh) and weight-average molecular weight (Mw), can be tuned up by controlling the electrolysis time, current density (j) and PVP and Fe2+ concentrations, as demonstrated via dynamic and static light scattering and gel permeation chromatography analysis. Using PVP at 0.25 wt.%, Fe2+ at 0.5-1.0 mmol dm-3 and low j, short treatment times induced intramolecular crosslinking with chain scission, allowing size reduction of PVP particles from 24 to 9-10 nm. Longer reaction times and higher PVP and Fe2+ contents favored intermolecular crosslinking ending in Mw values higher than the initial 3.95×105 g mol-1. An excessive ¿OH dose from a too high circulated charge (Q), i.e., too prolonged electrolysis time even at low j or too high j even for short time, promoted intramolecular crosslinking (Rh ~ 10-12 nm) along with a very significant chain scission probably owing to the loss of mobility of the three-dimensional nanogel network. In conclusion, EF allowed transforming the architecture of linear, inert PVP chains into a functionalized nanogel with -COOH and succinimide groups that have great potential for further conjugation

Facile crosslinking of poly(vinylpyrrolidone) by electro-oxidation with IrO2-based anode under potentiostatic conditions

The modification of polymer architectures by reaction with chemically adsorbed hydroxyl radicals has been thoroughly investigated by electrolyzing dilute aqueous solutions of the biocompatible polymer poly(vinylpyrrolidone) (PVP), using an undivided electrolytic cell with a Ti/IrO2-Ta2O5 (DSA®) anode. Several electrolyses were performed to assess the influence of the applied potential, the circulated charge and the PVP concentration, which was always kept low to avoid chain overlapping. From the results obtained, it can be concluded that the electro-oxidation of PVP solutions using a cheap anode is an effective method to crosslink initially isolated polymer chains, eventually increasing the size of their random coils. Furthermore, the average size of the modified macromolecules can be controlled by tuning the electrode potential and/or the current density and the circulated charge. At high anodic potential values, the hydroxyl radicals formed at DSA® were also effective to generate reactive functional groups on the PVP backbone, which is a very interesting feature for future biomedical applications

Crosslinking of poly(vinylpyrrolidone) activated by electrogenerated hydroxyl radicals: a first step towards a simple and cheap synthetic route of nanogel vectors

A facile electrosynthesis route for the preparation of polymer nanogels based on the in situ production of hydroxyl radicals is reported for the first time. Electro-Fenton process with continuous H2O2 electrogeneration and Fe2+ regeneration performs better than electro-oxidation with a boron-doped diamond or dimensionally stable anode for promoting crosslinking of poly(vinylpyrrolidone)

Water-borne Polymeric Nanoparticles for Glutathione-Mediated Intracellular Delivery of Anticancer Drugs.

A new family of water-borne, biocompatible and carboxyl- functionalized nanogels was developed for glutathione- mediated delivery of anticancer drugs. Poly(N-vinyl- pyrrolidone)-co-acrylic acid nanogels were generated by e- beam irradiation of aqueous solutions of a crosslinkable polymer, using industrial-type linear accelerators and set- ups. Nanogels physico-chemical properties and colloidal stability, in a wide pH range, were investigated. In vitro cell studies proved that the nanogels are fully biocompatible and able to quantitatively bypass cellular membrane. An anticancer drug, doxorubicin (DOX), was linked to the carboxyl groups of NGs through a spacer containing a disulphide cleavable linkage. In vitro release studies showed that glutathione is able to trigger the release of DOX through the reduction of the S-S linkage at a concentration comparable to its levels in the cytosol

Comparative studies of the Pschorr reaction in the pyrazole series. Access to the new dibenzo(e,g)pyrazolo(1,5-a)(1,3)diazocine system of pharmaceutical interest

The diazonium tetrafluoroborate 11 obtained from 2-amino-N-methyl-N-(1-phenyl-3-methylpyrazol-5-yl)benzamide was transformed in dry acetonitrile via an ionic or radical pathway. Diferences were observed with respect to ionic or radical transformations in aqueous media of the analogous diazonium hydrogen sulfate 1 derived from the same amine. In acetonitrile solution, the ionic pathway was characterized by an increased yield of 1,4-dimethyl-3-phenyl-pyrazolo[3,4-c]isoquinolin-5-one 4 and by the formation of its isomer, the new derivative 7,9-dimethyldibenzo[e,g]pyrazolo[1,5-a][1,3]diazocin-10(9H)-one 12. When the reaction folowed a radical pathway, the pyrazolo[3,4-c]isoquinoline derivative 4 and N-methyl-2-(1-phenyl-3-methylpyrazol-5-yl)benzamide 17, the later due to a 1,4-pyrazolyl transfer proces, were isolated in low yields. Decomposition of the solid diazonium tetrafluoroborate at its melting point gave compounds 4, 12 and the N-(1-phenyl-3-methylpyrazol-5-yl)-2-fluorobenzamide 17. The crystal structure of compound 12 was also determined

Gadolinium-chelating nanogels as MR contrast agesnts specifically targeting tumor cells

Development of multifunctional nanogels coordinating paramagnetic ions and displacing targeting ligands for preferential accumulation into tumors. Low molecular-weight Gd-chelates are widely used in clinical MRI for various purposes. However, these contrast agents (CAs) have several shortcomings: they rapidly extravasate from blood vessels to the interstitial space, have a short circulation times and show poor contrast at high magnetic fields. Incorporating gadolinium into flexible nanogels has the potential of increasing intravascular half-life, accumulation and retention in specific body compartments of the CA as well as increasing the MR signal, since many metal ions can be coordinated to the same nanoparticl