RESUMO
Thrombin is an essential part of the blood coagulation system; it is a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, and catalyzes many other coagulation-related reactions. Absorption at its surface of small nanoparticles can completely change the biological properties of thrombin. We have analyzed the influence on thrombin of 3 different kinds of small nanoparticles: dendrimers (phosphorus-based, carbosilane based and polyamidoamine) and 2 hybrid systems containing carbosilane, viologen and phosphorus dendritic scaffolds in one single molecule, bearing different flexibility, size and surface charge. There was significant alteration in the rigidity of the rigid dendrimers in contrast to flexible dendrimers. These differences in their action are important in understanding interactions taking place at a bio-nanointerface.
Assuntos
Dendrímeros/química , Nanopartículas/química , Fósforo/química , Silanos/química , Trombina/química , Sítios de Ligação , Coagulação Sanguínea , Humanos , Cinética , Modelos Moleculares , Nanopartículas/ultraestrutura , Nanotecnologia , Ligação Proteica , Estrutura Secundária de Proteína , Soluções , Eletricidade EstáticaRESUMO
This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.
Assuntos
Dendrímeros/química , Neoplasias/terapia , Fósforo/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Silanos/química , Transfecção/métodos , Apoptose , Sobrevivência Celular , Dicroísmo Circular , Dendrímeros/toxicidade , Eletroforese em Gel de Ágar , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Células HeLa , Heparina/química , Humanos , Luz , Microscopia Eletrônica de Transmissão , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Conformação de Ácido Nucleico , Tamanho da Partícula , Fósforo/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Espalhamento de Radiação , Silanos/toxicidade , Espectrometria de Fluorescência , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl-2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B).
Assuntos
Dendrímeros/química , Fósforo/química , RNA Interferente Pequeno/química , Terapêutica com RNAi/métodos , Silanos/química , Transfecção , Dicroísmo Circular , Eletroforese em Gel de Ágar , Regulação Neoplásica da Expressão Gênica , Heparina/química , Humanos , Luz , Microscopia Eletrônica de Transmissão , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Conformação de Ácido Nucleico , Tamanho da Partícula , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espalhamento de Radiação , Espectrometria de Fluorescência , Transfecção/métodos , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The development of medical nanosystems requires knowledge of their behavior in vivo. Clinical chemistry tests are widely used to estimate the systemic toxicity of nanoparticles. In this paper we have explored the impact of small positively charged nanoparticles-poly(amidoamine), phosphorous and carbosilane dendrimers - on biochemical parameters of standardized serum in vitro. All the dendrimers could shift the main biochemical parameters. Thus, in the case of patients having the normal, but 'boundary', values of biochemical parameters, nanoparticle-induced changes can be wrongly interpreted as evidence of some dysfunctions (hepatic, renal, etc.). Moreover, the effects of nanoparticles of metals, carbon nanotubes, quantum dots, fullerenes, dendrimers having been sized up to 4000 nm and the hundreds of reactive groups, can be significantly higher. Thus, preliminary evaluation of any nanomaterial in vitro is required in clinical chemistry tests before its application in vivo to draw the correct conclusions and benefit animals.