RESUMO
The aim of this work was to investigate novel formulations containing diruthenium(II-III)-ibuprofen (RuIbp) metallodrug encapsulated into the chitosan (CT) biopolymer. Microparticles (RuIbp/CT MPs, â¼ 1 µm) were prepared by spray-drying, and RuIbp/CT-crosslinked nanoparticles (NPs) by ionic gelation (RuIbp/CT-TPP, TPP = tripolyphosphate (1), RuIbp/CT-TPP-PEG, PEG = poly(ethyleneglycol (2)) or pre-gel/polyelectrolyte complex method (RuIbp/CT-ALG, ALG = alginate (3)). Ru analysis was conducted by energy dispersive x-ray fluorescence or inductively coupled plasma atomic emission spectroscopy, and physicochemical characterisation by powder x-ray diffraction, electronic absorption and FTIR spectroscopies, electrospray ionisation mass spectrometry, thermal analysis, scanning electron, transition electron and atomic force microscopies, and dynamic light scattering. The RuIbp-loaded nanosystems exhibited encapsulation efficiency â¼ 20-37%, drug loadingâ¼ 10-20% (w/w), hydrodynamic diameter (nm): 103.2 ± 7.9 (1), 91.7 ± 12.6 (2), 270.2 ± 58.4 (3), zeta potential (mV): +(47.7 ± 2.8) (1), +(49.2 ± 3.6) (2), -(28.2 ± 2.0) (3). Nanoformulation (1) showed the highest cytotoxicity with increased efficacy in relation to the RuIbp free metallodrug against U87MG human glioma cells.
RESUMO
The objectives of this work are to develop nanocarrier systems for the Ru(II)-p-cymene naproxen antitumor metallodrug, [Ru(η6-p-cymene)(npx)Cl] or Rupcy, based on polymeric nanoparticles (NPs) composed by the biodegradable poly(lactic acid) (PLA) and the hydrophilic polymerised ß-cyclodextrin (PolyCD); to validate an analytical method for determination of Ru incorporated into the metallodrug loaded-NPs. The PolyCD was prepared by single step condensation and polymerisation reaction and incorporated as a polymer blend during the fabrication of PLA/PolyCD blends NPs and also as a core/shell structure built by adsorption of the PolyCD onto the surface of PLA NPs to give PLA(core)/PolyCD(shell) NPs. Three different loaded-systems incorporating the metallodrug (Rupcy-PLA NPs (1), Rupcy-PLA/PolyCD blends (2), and Rupcy-PLA(core)/PolyCD(shell) NPs (3)) were prepared by nanoprecipitation. The characterisation was performed by Proton Nuclear Magnetic Resonance, Matrix Assisted Laser Desorption/Ionization Time-of-Flight, Fourier-Transform Infra-red and UV-VIS Electronic Absorption Spectroscopies, Thermogravimetric Analysis, Differential Scanning Calorimetry, Dynamic Light Scattering, and Electrophoretic Light Scattering. Ru was determined by Microwave Induced Plasma Optical Emission Spectrometry (MIP-OES) with validation of the method. The metallodrug entrapment efficiency was around 90% (w/w) and drug loading was at 3-4% (w/w). The characterised metallodrug-loaded systems exhibited monomodal size distributions and appropriate hydrodynamic diameters [218.3 ± 13.5 (1), 205.4 ± 14.4 (2), 231.5 ± 22.0 (3) nm] and zeta potential values [-31.5 ± 2.2 (1), -26.1 ± 4.5 (2), -28.8 ± 6.1 (3) mV]. The validation of the MIP-OES method by evaluating selectivity, linearity, precision, accuracy, and limits of detection and quantification succeeded. The NPs parameters are compatible with colloidally stable systems. The MIP-OES method showed to be simple, reliable, and feasible to quantify indirectly the amount of the metallodrug-loaded into the PLA NPs.
Assuntos
Nanopartículas , Rutênio , Naproxeno , Micro-Ondas , Poliésteres/química , Polímeros/química , Nanopartículas/química , Análise Espectral , Tamanho da Partícula , Portadores de Fármacos/químicaRESUMO
Diruthenium(II,III) metal-metal multiply bonded paddlewheel complexes bearing non-steroidal anti-inflammatory drugs are promising anticancer metallodrugs. The [Ru2(Ibp)4Cl] (Ibp, ibuprofenate anion from HIbp ibuprofen drug), free or encapsulated, shows anticancer activity against glioblastoma (in vitro, in vivo), and against human breast and prostate cancer cells. Herein we report the interaction of [Ru2(Ibp)4Cl] and of [Ru2(Ac)4(H2O)2]PF6 (Ac, acetate) with the 4-aminopyridine (4Apy) drug. The N-ligand was capable of cleaving the paddlewheel unit with oxidation of Ru2(II,III) to Ru2(III,III)O µ-oxo core in the ibuprofen complex while the acetate complex underwent axial substitution of water by 4Apy. Carefully designed synthetic and chromatographic methods succeeded in giving the novel [Ru2O(Ibp)2(4Apy)6]Cl2 metallodrug, the first diruthenium(III,III) µ-oxo having chloride as counterion. Characterization was performed by elemental analysis, mass spectrometry, thermogravimetric analysis, electronic absorption and vibrational spectroscopies, molar conductivity and cyclic voltammetry. Kinetic studies for the µ-oxo complex (in 50:50 v/v ethanol:water) suggested an aquation/complexation equilibrium in consecutive step reactions with the exchange of the two 4Apy trans to the µ-oxo bridge by water (aquation) and the back coordination of 4Apy in excess of the N-ligand (complexation). Trypan blue assays for the novel compound showed time- and dose- dependent antiproliferative effects (at 5-50 µmol L-1) and cytotoxicity (> 20 µmol L-1), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assays gave IC50 value of 7.6 ± 1.5 µmol L-1 (at 48 h, 1-20 µmol L-1) against U87MG human glioblastoma cells (aggressive brain glioma cancer) pointing the metallodrug as potential candidate for novel therapies in gliomas.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ibuprofeno/análogos & derivados , Ibuprofeno/farmacologia , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Rutênio/químicaRESUMO
A unique class of diruthenium(ii,iii) metallodrugs containing non-steroidal anti-inflammatory drug (NSAID), Ru2(NSAID), have been reported to show anticancer activity in glioma models in vitro and in vivo. This work reports the encapsulation of the lead metallodrug of ibuprofen (HIbp), [Ru2(Ibp)4Cl] or RuIbp, and also of the new analogue of naproxen (HNpx), [Ru2(Npx)4Cl] or RuNpx, in novel intravenously (i.v.) injectable solid polymer-lipid nanoparticles (SPLNs). A rationally selected composition of lipids/polymers rendered nearly spherical Ru2(NSAID)-SPLNs with a mean size of 120 nm and zeta potential of about -20 mV. The Ru2(NSAID)-SPLNs are characterized by spectroscopic techniques and the composition in terms of ruthenium-drug species is analyzed by mass spectrometry. The metallodrug-loaded nanoparticles showed high drug loading (17-18%) with â¼100% drug loading efficiency, and good colloidal stability in serum at body temperature. Fluorescence-labeled SPLNs were taken up by the cancer cells in a time- and energy-dependent manner as analyzed by confocal microscopy and fluorescence spectrometry. The Ru2(NSAID)-SPLNs showed enhanced cytotoxicity (IC50 at 60-100 µmol L-1 ) in relation to the corresponding Ru2(NSAID) metallodrugs in breast (EMT6 and MDA-MB-231) and prostate (DU145) cancer cells in vitro. The cell viability of both metallodrug nanoformulations is also compared with those of the parent NSAIDs, HIbp and HNpx, and their corresponding NSAID-SPLNs. In vivo and ex vivo fluorescence imaging revealed good biodistribution and high tumor accumulation of fluorescence-labeled SPLNs following i.v. injection in an orthotopic breast tumor model. The enhanced anticancer activity of the metallodrug-loaded SPLNs in these cell lines can be associated with the advantages of the nanoformulations, assigned mainly to the stability of the colloidal nanoparticles suitable for i.v. injection and enhanced cellular uptake. The findings of this work encourage future in vivo efficacy studies to further exploit the potential of the novel Ru2(NSAID)-SPLN nanoformulations for clinical application.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos , Ibuprofeno/administração & dosagem , Lipídeos , Nanopartículas , Naproxeno/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Ibuprofeno/farmacologia , Masculino , Naproxeno/farmacologia , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/farmacologia , Polímeros , Rutênio , Distribuição TecidualRESUMO
The metallodrug chloridotetrakis(ibuprofenato)diruthenium(II,III) ([Ru2(Ibp)4Cl] or RuIbpCl (1), Ibp=carboxylate anion derived from the non-steroidal anti-inflammatory drug ibuprofen) has shown promising results in vitro and in vivo, which point to its potential as an inhibitor of glioma tumour growth in vivo. In order to get insight into the influence of structural changes on the biological response of the metallodrug, the [Ru2(Ibp)4] metal-metal multiply bonded paddlewheel unit was modified for the axial ligand. Two new analogues, [Ru2(Ibp)4(CF3SO3)] (2) and [Ru2(Ibp)4(EtOH)2]PF6 (3), were synthesized and fully characterized by elemental analysis, ESI-MS, vibrational (FTIR, Raman) and electronic (UV/VIS/NIR) spectroscopy, magnetic susceptibility, molar conductivity measurements, and thermal analysis. RuIbpCl was re-prepared and complementary characterization to previous work was performed. The three axially-modified RuIbp metallodrugs were compared for their effects on U87MG and A172 human glioma cell proliferation, apoptosis, mitosis, and cell migration in vitro. The results provide evidence that the chloride ligand in RuIbpCl may play key role in the mode of action of the metallodrug, since the best results for antiproliferative activity were found for (1) in both types of human glioma cells. All the metallodrugs, (1), (2) and (3), were uptaken by the cells, and were shown to cause increase on number of apoptotic cells and decrease on number of mitotic cells. Additionally, the RuIbp metallodrugs were capable of inhibiting cell migration process in both human glioma cell lines. These data are extremely promising as drugs which can inhibit both cell proliferation/mitosis and inhibit cell migration could target two major chemotherapeutic targets in high grade gliomas.
Assuntos
Antineoplásicos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glioma/tratamento farmacológico , Ibuprofeno , Mitose/efeitos dos fármacos , Compostos Organometálicos , Rutênio , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Glioma/metabolismo , Humanos , Ibuprofeno/química , Ibuprofeno/farmacologia , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Rutênio/química , Rutênio/farmacologiaRESUMO
The Diruthenium-Ibuprofen compound [Ru2Cl(Ibp)4] (or RuIbp) is known to cause significant inhibition of C6 rat glioma cell proliferation in vitro. RuIbp increased the expression of cell cycle-related proteins such as p21 and p27 and the pro-apoptotic protein Bax, as well as causing a reduction in mitochondrial membrane potential and a modest increase in apoptosis in vitro. The present study extended these findings by (i) investigating the effects of RuIbp on human glioma cell line proliferation in vitro and (ii) investigating the acute and chronic toxicology of the compound in normal Wistar rats. The compound was then tested for its anti-tumour properties by either chronic 14 days intra-peritoneal (IP) administration or chronic Alzet osmotic pump infusion, in the rat C6 orthotopic glioma model in vivo. The IP injection of RuIbp caused a 41 % inhibition of tumour area without significant toxic effects but with an increase in blood neutrophils and monocytes and a decrease in blood lymphocytes. In an attempt to reduce this effect RuIbp was administered by Alzet osmotic pump infusion directly into the tumour at a dose of 15 mg/kg with an infusion rate of 0.5 µL/h for 14 days. The direct infusion of RuIbp caused a 45 % inhibition of tumour area without alterations in differential blood leukocyte counts. These results prove the efficacy of RuIbp in human glioma cell lines in vitro and in an in vivo glioma model and point to its potential as an inhibitor of tumour growth in vivo.
Assuntos
Antineoplásicos/farmacologia , Glioma/tratamento farmacológico , Glioma/patologia , Ibuprofeno/química , Neoplasias Experimentais/tratamento farmacológico , Compostos Organometálicos/farmacologia , Rutênio/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Neoplasias Experimentais/patologia , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/química , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
AIM: Gliomas are primary brain tumours. Gamma-linolenic acid (GLA) exerts anti-proliferative effects. Several ruthenium-containing complexes have antiproliferative effects and can be used as adjuvant therapies in cisplatin-resistant cancer. The present study reports on the anti-proliferative properties and effects on tumour morphology of a novel diruthenium-GLA complex (Ru2GLA) and its comparison with GLA in the C6 rat glioma model both in vitro and in vivo. MATERIALS AND METHODS: In vitro and in vivo experiments were performed on C6 glioma rat cells, and in an orthotopic model. RESULTS: Ru2GLA (100 µM) appears to be an inhibitor of C6 rat glioma cell proliferation. The nuclear area of Ru2GLA-treated cells was 2.18-times larger than that of control cells, suggesting DNA replication occurred but mitosis was blocked in the G2-M phase. Ru2GLA (2 mM) inhibited C6 cell proliferation in vivo and the changes in tumor morphology confirm both cellular uptake and collagen fibre-binding in the extracellular matrix. CONCLUSION: Ru2GLA appears to be a low-toxicity drug and a potential candidate for anti-proliferative therapy of glioma.
Assuntos
Glioma/patologia , Rutênio/farmacologia , Ácido gama-Linolênico/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Glioma/tratamento farmacológico , Glioma/ultraestrutura , Ratos , Rutênio/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Ácido gama-Linolênico/administração & dosagemRESUMO
Diruthenium(ii,iii)-tetracarboxylates have shown promising anticancer properties as metallotherapeutics. On the basis of the role that bio-reducing agents may play on the mode of action of ruthenium-based anticancer drugs, we performed detailed kinetic studies on the reaction of ascorbic acid and glutathione with the [Ru2(RCOO)4](+) paddlewheel framework by using the non-drug, diaqua complex ion [Ru2(CH3COO)4(H2O)2](+). In the presence of the reducing agents, the diaqua-Ru2 species first undergo a ligand substitution reaction by which the axially-coordinated water is displaced by the reducing agent. In both cases, this reaction is followed by an intra-molecular electron transfer process during which the metal-metal center is reduced from Ru2(5+) to Ru2(4+) and the reducing agent is oxidized. Product analyses were performed with the application of ESI-MS and (1)H-NMR techniques. Rate and activation parameters are reported for the different reaction steps.
Assuntos
Ácido Ascórbico/metabolismo , Ácidos Carboxílicos/farmacologia , Glutationa/metabolismo , Substâncias Redutoras/metabolismo , Rutênio/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Ácidos Carboxílicos/química , Cinética , Rutênio/químicaRESUMO
The known paddlewheel, tetrakis(acetato)chloridodiruthenium(II,III), offers a versatile synthetic route to a novel class of antitumor diruthenium(II,III) metallo drugs, where the equatorial ligands are nonsteroidal anti-inflammatory carboxylates. This complex was studied here as a soluble starting prototype model for antitumor analogues to elucidate the reactivity of the [Ru(2)(CH(3)COO)(4)](+) framework. Thermodynamic studies on equilibration reactions for axial substitution of water by chloride and kinetic studies on reactions of the diaqua complexes with the amino acids glycine, cysteine, histidine, and tryptophan were performed. The standard thermodynamic reaction parameters ΔH°, ΔS°, and ΔV° were determined and showed that both of the sequential axial substitution reactions are enthalpy driven. Kinetic rate laws and rate constants were determined for the axial substitution reactions of coordinated water by the amino acids that gave the corresponding aqua(amino acid)-Ru(2) substituted species. The results revealed that the [Ru(2)(CH(3)COO)(4)](+) paddlewheel framework remained stable during the axial ligand substitution reactions and was also mostly preserved in the presence of the amino acids.
Assuntos
Aminoácidos/química , Compostos Organometálicos/química , Rutênio/química , Termodinâmica , Cinética , Compostos Organometálicos/síntese químicaRESUMO
The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)4Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 microM at 24 h, 211 microM at 48 h to 81 microM at 72 h. In conclusion, Ru(2)GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Glioma/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Ácido gama-Linolênico/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Fase G1 , Glioma/metabolismo , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Oxazinas/farmacologia , Ratos , Espectrofotometria Atômica , Coloração e RotulagemRESUMO
The immobilization of the NSAID ibuprofen (Hibp) and the Cu(II)-ibp compound on magnesium-aluminum layered double hydroxides (Mg3Al-LDH) is described. Ibuprofen was intercalated on LDHs by three routes (ion exchange, co-precipitation, and reconstruction). The organic drug and the Cu(II)-ibp were also immobilized by adsorption on LDH external surfaces. Materials were characterized by elemental analysis, UV/VIS, FTIR, and Raman spectroscopies, powder X-ray diffractometry (XRD), thermogravimetry, and electronic paramagnetic resonance (EPR). Mg3Al-(ibp)(cop) (30% w/w of drug/material) and Mg3Al-(ibp)(ie) (33%) materials exhibit bilayer arrangements of ibp anions intercalated between the host layers. Mg3Al-(ibp)(rec) and Mg3Al-(ibp)(ads) contain 13% and 6.2% of ibuprofenate, respectively. Mg3Al-(Cu-ibp)(ads) exhibits two Cu centers in different environments interacting with host layers. Pharmacological potential of materials are compared considering the amounts of immobilized drugs and their buffering properties. Mg3Al-(ibp)(ie) and Mg3Al-(ibp)(cop) exhibit poor buffering property, but contain high ibp amounts. Mg3Al-(ibp)(ads) despite having buffering property, contains low amount of ibuprofen. Mg3Al-(ibp)(rec) combines significant amount of immobilized ibp with good buffering property. Mg3Al-(Cu-ibp)(ads), due to the buffering property, may be an interesting new formulation aiming to decrease gastric irritation.