Guendalina Zuccari

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

BackgroundNeuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred.ResultsWe could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells.ConclusionSodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents.

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.

Hydrogels Formed by Crosslinked Poly(vinyl alcohol) as Sustained Drug Delivery Systems.

Poly(vinyl alcohol) was crosslinked with ethylene glycol diglycidyl ether to obtain hydrogel‐forming polymers. The polymers were also substituted with oleoyl chloride, providing hydrogels with weak solubility.These new polymeric materials were evaluated for the formulation of sustained drug delivery systems. Vancomycin hydrochloride was used as a peptidic model drug whose sustained release should minimize its inactivation in the upper part of the gastrointestinal tract. Spray‐dried mixtures of the drug and the polymer [at 1:4 and 1:8 (w:w) ratios] were prepared and the release of the drugs from the mixtures was evaluated in vitro at pH 2.0, 5.5, 7.4, and 8.0. The results indicated that the crosslinked polymers slowed down the release of the drugs with respect to the pure drug at each pH.The degree of crosslinking of ethylene glycol diglycidyl ether and the extent of substitution with oleoyl chloride were found to influence drug release.

Tumor vascular targeted liposomal-bortezomib minimizes side effects and increases therapeutic activity in human neuroblastoma.

Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies.

Polyvinylalcohol substituted with triethyleneglycolmonoethylether as a new material for preparation of solid dispersions of hydrophobic drugs

Among the different methods used to increase the aqueous drug solubility, the preparation of a solid dispersion with a soluble carrier represents an interesting formulative approach. We substituted polyvinylalcohol with triethyleneglycolmonoethylether and obtained a suitable material for the formulation of a solid dispersion of progesterone, by spray-drying. In particular, we evaluated the influence of the polyvinylalcohol substitution degree and the polymer-drug weight ratios in the preparative mixture on the progesterone dissolution rate in the aqueous environment.

Poly(Vinylalcohol-Co-Vinyloleate) for the Preparation of Micelles Enhancing Retinyl Palmitate Transcutaneous Permeation

The amphiphilic properties of poly(vinylalcohol) substituted with oleic acid was evaluated to assess the possibility to prepare polymeric micelles in an aqueous phase containing a hydrophobic core able to host lipophilic drugs such as retinyl palmitate and thereby enhance its transcutaneous absorption in the stratum corneum. The effect of the increased drug absorption suggests the possibility of interaction between the substituted polymer and the components present in the intercorneocyte spaces. Correlations between the drug concentration in the preparative mixture, micelle size, and drug permeation were evaluated to establish the best functional properties of the micellar systems enhancing retinyl palmitate absorption. Transcutaneous absorption increased with decreasing micelle size, and micelle size decreased on decreasing the drug concentration in the preparative mixture.

Enhanced anti-tumor and anti-angiogenic efficacy of a novel liposomal fenretinide on human neuroblastoma

Neuroblastoma is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest. It approximately accounts for about 15% of all pediatric oncology deaths. Despite advances in multimodal therapy, metastatic neuroblastoma tumors at diagnosis remain a clinical challenge. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of neuroblastoma cells. Among them, fenretinide (HPR) has been considered one of the most promising anti-tumor agent but it is partially efficacious due to both poor aqueous solubility and rapid metabolism. Here, we have developed a novel HPR formulation, by which the drug was encapsulated into sterically stabilized nanoliposomes (NL[HPR]) according to the Reverse Phase Evaporation method. This procedure led to a higher structural integrity of liposomes in organic fluids for a longer period of time, in comparison with our previous liposomal formulation developed by the film method. Moreover, NL[HPR] were further coupled with NGR peptides for targeting the tumor endothelial cell marker, aminopeptidase N (NGR-NL[HPR]). Orthotopically xenografted neuroblastoma-bearing mice treated with NGR-NL[HPR] lived statistically longer than mice untreated or treated with free HPR (NGR-NL[HPR] vs both control and HPR: P<0.0001). Also, NL[HPR] resulted in a statistically improved survival (NL[HPR] vs both control and HPR: P<0.001) but to a less extent if compared with that obtained with NGR-NL[HPR] (NGR-NL[HPR] vs NL[HPR]: P<0.01). Staining of tumor sections with antibodies specific for neuroblastoma and for either pericytes or endothelial cells evidenced that HPR reduced neuroblastoma growth through both anti-tumor and anti-angiogenic effects, mainly when delivered by NGR-NL[HPR]. Indeed, in this group of mice a marked reduction of tumor progression, of intra-tumoral vessel counts and VEGF expression, together with a marked down-modulation of matrix metalloproteinases MMP2 and MMP9, was observed. In conclusion, the use of this novel targeted delivery system for the apoptotic and antiangiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of neuroblastoma patients.

Novel micelles based on amphiphilic branched PEG as carriers for fenretinide

This study reports on the preparation and evaluation of amphiphilic macromolecules based on branched polyethylene glycol covalently linked with alkyl hydrocarbon chains. These macromolecules easily dissolved in an aqueous environment, with formation of micellar nanoaggregates endowed with hydrophobic inner cores capable of hosting fenretinide by complexation. The complexes increased fenretinide aqueous solubility, while hindering its release as a free drug in an aqueous environment. Particle size analysis indicated dimensional suitability of the complexes for intravenous administration. Neuroblastoma cell lines (SH-SY5Y and NGP) exhibited increased sensitivity to fenretinide in complex as compared to free drug, associated with higher intracellular concentrations of fenretinide observed after treatment with the complex. Transmission electronic microscopy images revealed endocytosis of the micellar complex. Moreover, fenretinide conversion to its metabolite 4-oxo-fenretinide was delayed in cells treated with the complex, further supporting the hypothesis that fenretinide may be absorbed by micellar transport and exposed to the cytoplasm for conversion to its metabolite only after micelle destabilization.

Modified Doxorubicin for Improved Encapsulation in PVA Polymeric Micelles

Polyvinylalcohol, partially substituted with lipophilic acyl chains, generates polymeric micelles in aqueous phase, containing a hydrophobic core able to encapsulate lipophilic drugs. Two types of polymers were obtained by conjugation of polyvinylalcohol with oleoyl or linoleoyl chains as pendant groups. The polymers, at a substitution degree of ∼1%, are soluble in water and form polymeric micelles whose size increases with polymer concentration. Doxorubicin was hydrophobized, by linking an oleoyl chain via amide bond, to make the drug more similar to the substituted polymers and promote its encapsulation into the inner core of the micelles. The properties of the drug-polymer systems were evaluated in solution by dynamic light scattering technique and correlated to the physicochemical characteristics of the drug and the substituted polymers. Solubilization tests revealed that the similarity of the chain, in both the polymer and the drug, promotes better drug encapsulation in the oleoyl than linoleoyl derivative. The drug-polymer systems are stable in phosphate buffer saline (pH 7.4) at 37°C, and the release of the drug is activated by the presence of the proteolytic enzyme pronase-E. The enzyme activated drug release and the size of the polymeric micelles, compatible with the pore dimensions of the tumor vessels, make these systems interesting for targeting lipophilic drugs to solid tumors, where the proteolytic enzyme concentration strongly raises with respect to the other body compartments.

In vitro and In vivo Antitumor Activity of the Novel Derivatized Polyvinyl Alcohol-Based Polymer P10(4)

Purpose: The major limitation to successful chemotherapy of neuroblastoma is the toxicity of traditional antitumor drugs. Hence, less toxic and more effective drugs are to be found, and novel formulations of conventional compounds allowing a more favorable biodistribution should be sought for. In an attempt to pursue this task, we recently synthesized an amphiphilic polymer based on a polyvinyl alcohol backbone [P10(4)]. Experimental Design: The cytotoxic activity of P10(4) was evaluated both in vitro on neuroblastoma and melanoma cell lines and in vivo in pseudometastatic neuroblastoma models. Apoptosis was assessed by morphology, cytofluorimetric analysis of DNA content, and DNA fragmentation assay. Caspases activation was investigated by kits specific for caspase-1, caspase-2, caspase-3, caspase-4, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and caspase-13. Colony formation was evaluated by soft agar assay. Results: P10(4) exerted a potent cytotoxic activity on different neuroblastoma and melanoma cell lines through induction of both extrinsic and intrinsic caspase cascades and subsequent apoptosis. Moreover, the clonogenic potential of cells that survived P10(4) treatment was strongly reduced. Next, we tested the effects of P10(4) in nude mice injected with both a human and a murine neuroblastoma cell lines i.v. P10(4) significantly increased the life span and the long-term survival of treated mice over controls. No side effects were observed, even at doses higher than those used for therapeutic purposes. Conclusions: Our data suggest that P10(4) holds promise as an anticancer compound and, because of its lack of interaction with DNA, is unlikely to give rise to drug resistance.