RESUMO
Branched polyethylenimine (PEI, 25 kDa) was ionically interacted with varying amount of alginic acid to block different proportion (2.6-5.7%) of amines in PEI to form a series of nanocomposites, PEI-Al. These nanocomposites, upon interaction with DNA, protected it against DNase I. Among various complexes evaluated, PEI-Al(4.8%)/DNA displayed the highest transfection efficiency in HEK293, COS-1 and HeLa cells that was approximately 2-8-folds higher than Superfect, Fugene, PEI (750 kDa)-Al(6.26%) and PEI alone. The projected nanocomposites were nearly non-toxic to cells in vitro. Furthermore, the concentration of PEI-Al(4.8%) needed to deliver GFP-specific siRNA in COS-1 cells was 20 times lower than PEI (750 kDa)-Al(6.26%). Intracellular trafficking of PEI-Al(4.8%) with or without complexed DNA in HeLa cells shows that both appear in the nucleus after 1 h.
Assuntos
Alginatos/química , Núcleo Celular/metabolismo , DNA/metabolismo , Nanopartículas , Polietilenoimina/química , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Transporte Ativo do Núcleo Celular , Alginatos/toxicidade , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , DNA/química , Desoxirribonuclease I/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/toxicidade , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HeLa , Ácidos Hexurônicos/química , Ácidos Hexurônicos/toxicidade , Humanos , Polietilenoimina/toxicidade , Interferência de RNA , RNA Interferente Pequeno/química , Fatores de TempoRESUMO
Polyethylenimine (PEI), a widely used cationic polymeric vector with high transfection efficiency, was converted into nanoparticles by introducing ionic and covalent crosslinkers with varying proportion of 1,6-hexanebisphosphate (HP), adipic acid (AA) and 1,4-butane dialdehyde (BA) to obtain a small library of HP-PEI (HPP), AA-PEI (AAP) and BA-PEI (BAP) nanoparticles, respectively. Particles were characterized by spectroscopic technique as well as physicochemical parameters such as size, morphology, surface charge, effect of crosslinking on buffering capacity and DNA binding ability. The entire series of nanoparticles were compared for their cytoxicity and ability to deliver genes in various cell lines. Among various nanoparticles, AAP-3 nanoparticle/DNA complex exhibited higher transfection efficiency (1.5-7.8 folds) than the native PEI (25kDa) and commercially available transfection reagents, such as GenePorter, GenePorter2, Fugene and Superfect, with cell viability >85%. The highest cell viability was observed with BAP nanoparticles (>95%). Importantly, the transfection activity of nanoparticle/DNA complexes was preserved in the presence of serum. Transfection with GFP-siRNA inhibited expression of transfected GFP gene by approximately 81-92%. All nanoparticle types (HPP, AAP and BAP) required a comparable time for entry into cells and subsequent intracellular passage from the cytoplasm to the nucleus. Intravenous delivery of (99)Tc labeled BAP-2/DNA complex to female Balb/c mice revealed the presence of the complex in most of the organs with the highest retention in liver. In conclusion, HPP, AAP and BAP nanoparticles are safe for efficient gene delivery.
Assuntos
DNA/administração & dosagem , Nanopartículas , Polietilenoimina/química , RNA Interferente Pequeno/administração & dosagem , Adipatos/química , Aldeídos/química , Animais , Linhagem Celular , Sobrevivência Celular , Reagentes de Ligações Cruzadas/química , DNA/farmacocinética , Feminino , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organofosforados/química , Tamanho da Partícula , RNA Interferente Pequeno/farmacocinética , Propriedades de Superfície , Distribuição Tecidual , Transfecção/métodosRESUMO
Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.