RESUMO
Chitosan scaffolds of different deacetylation degrees, average molecular weights and concentrations reinforced with silica nanoparticles were prepared for bone tissue regeneration. The resulting nanocomposites showed similar pore sizes (<300 µm) regardless the deacetylation degree and concentration used in their formulation. Their mechanical compression resistance was increased by a 30% with the addition of silica nanoparticles as nanofillers. The biocompatibility of the three-dimensional chitosan scaffolds was confirmed by the Alamar Blue assay in human primary osteoblasts as well as the formation of cell spheroids indicative of their great potential for bone regeneration. In vivo implantation of the scaffolds in a mice calvaria defect model provided substantial evidences of the suitability of these nanocomposites for bone tissue engineering showing a mature and dense collagenous tissue with small foci of mineralization, vascularized areas and the infiltration of osteoblasts and osteoclasts. Nevertheless, mature bone tissue formation was not observed after eight weeks of implantation.
Assuntos
Regeneração Óssea , Quitosana/análogos & derivados , Nanocompostos/química , Osteogênese , Dióxido de Silício/química , Alicerces Teciduais/química , Animais , Substitutos Ósseos/química , Células Cultivadas , Humanos , Masculino , Teste de Materiais , Camundongos Nus , Nanocompostos/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Osteoblastos/citologia , Porosidade , Crânio/lesões , Crânio/fisiologia , Engenharia TecidualRESUMO
The cytotoxic effect of iodide or thiocyanate copper(i) complexes (1-PSf, 2-PSf, 3-PSf, 4-PSf) with phosphine derived from sparfloxacin (HSf) and 2,9-dimethyl-1,10-phenanthroline (dmp) or 2,2'-biquinoline (bq) as diimine auxiliary ligands was proved in vitro on somatic (MRC-5) and neoplastic (MCF7) human cell lines. Differences in mode of action were investigated in-depth for the selected dmp and bq complexes (1-PSf, 3-PSf, respectively) by elucidation of the following: (i) the efficiency to produce reactive oxygen species (ROS) in biological systems (cyclic voltammetry); (ii) their impact on mitochondrial membrane potential; (iii) potency for the activation of caspases 3 and 9; (iv) influence on the degree of DNA degradation (comet assay). It was concluded that the apoptosis of cancer cells is directly connected to the caspase-dependent mitochondrial pathway and supported by ROS production along with irreversible DNA fragmentation. Finally, it was demonstrated that the selected copper(i) complex encapsulated inside liposomes (1-PSf-L) exhibited enhanced accumulation inside cancer cells. This resulted in its higher cytotoxicity against cancer cells with therapeutic index of ca. 60. Increased selective accumulation in active neoplasm with simultaneous enhanced bioavailability and reduced systemic toxicity of liposomal formulation of copper(i) complexes can result in the development of new copper-based therapeutics and their successful implementation in anticancer chemotherapy.
Assuntos
Morte Celular/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Fluoroquinolonas/química , Fosfinas/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Complexos de Coordenação/administração & dosagem , Complexos de Coordenação/metabolismo , Eletroquímica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Lipossomos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
The copper(ii) ion binding of the Ac-KGHGNG-NH2 and Ac-PTVHNE-NH2 fragments of FomA adhesin from Fusobacterium nucleatum was studied using potentiometry, UV-Vis, CD, EPR and DFT techniques. The coordination pattern was described in a wide range of pH values. Ligands begin interactions with metal ions using imidazole nitrogen. At pH 6.8 (a value typical of the large intestine environment), the metal ion was coordinated by the 3N donor atoms {Nim, 2 × N-amide} in both cases. However, the copper(ii) ion was bound more effectively by the Ac-PTVHNE-NH2 peptide. The formation of reactive oxygen species (ROS) was studied by UV-Vis and fluorescence spectroscopy, as well as gel electrophoresis in the presence of H2O2 and/or ascorbic acid. The complexes generated ROS in the highest amounts among all compounds. Moreover, they stimulated the CT26 cell line (mouse colon carcinoma) to produce ROS which lead to oxidative stress. It was also determined that such radicals took part in the plasmid degradation mechanism.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Neoplasias Colorretais/metabolismo , Complexos de Coordenação/farmacologia , Cobre/química , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Animais , Ácido Ascórbico/química , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Humanos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Imidazóis , Ligantes , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/químicaRESUMO
In this paper we present a first insight into the cytotoxic action mode of copper(I) iodide or copper(I) thiocyanate complexes with a phosphine derivative of sparfloxacin (a 3rd generation fluoroquinolone antibiotic agent) and 2,9-dimethyl-1,10-phenanthroline or 2,2'-biquinoline as auxiliary ligands. The in vitro cytotoxic activity of the new complexes was tested against two cancer cell lines (CT26--mouse colon carcinoma and A549--human lung adenocarcinoma). An ICP-MS study revealed a marked time-dependent intracellular copper accumulation of the tested compounds. In addition, confocal microscopy imaging showed accumulation of the complexes inside whole cells and their emission of blue light. The complexes generate reactive oxygen species in the cancer cells, which was examined by using two different fluorescent probes. Moreover, (I) DNA intercalation studied by luminescence spectroscopy, circular dichroism and molecular docking, and (II) plasmid DNA damage also demonstrate their significant cytotoxicity. All these observed biological effects contribute to the induction of apoptosis, observed at a great predominance.