RESUMO
OBJECTIVES: Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications. RESULTS: Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg(-1) silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg(-1) silica nanoparticles. CONCLUSIONS: Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.
Assuntos
Portadores de Fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Imobilizadas/metabolismo , Nanopartículas , Dióxido de Silício , Adsorção , Animais , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Clonagem Molecular , Difusão Dinâmica da Luz , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/isolamento & purificação , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Peso Molecular , Células RAW 264.7 , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria InfravermelhoRESUMO
Most of the cancer treatment strategies from chemotherapy to radiotherapy render cancer cells apoptotic and these apoptotic cancer cells accumulate at the tumor sites. The accumulation of apoptotic cancer cells often result in inflammation and autoimmune responses causing serious health implications. Macrophages, which are effective immune combatants, can help in the clearance of these deleterious occupants. Granulocyte macrophage colony stimulating factor (GM-CSF) is a key cytokine, modulator of immune system and responsible for growth and differentiation of granulocytes and macrophages. In this regard, supply of recombinant GM-CSF can enhance the capability of macrophages for clearance of apoptotic cancer cells. However, delivery of the cytokine in vivo can suffer from certain disadvantages like faster depletion, less stability and low targeting efficiency. We believe that the stability and sustained release of GM-CSF can be improved through its encapsulation inside appropriately designed nanoparticles.