Studies On Interferon-alpha PLGA Microspheres

Interferon-alpha(IFNα)was chosen as a model drug and poly(lactic acid-glycolic acid) (PLGA)was used as the carrier to prepare Interferon-alpha sustained-release microspheres by W/O/W double emulsions solvent evaporation method.Furthermore,the drug release behaviors and the properties of IFNαmicrospheres were deeply studied.During the process of IFNαmicrospheres preparation,the effect of various factors were studied at first with the particle's size and roudness of the microspheres as the criteria.Then the optimum formulation of IFNαmicrospheres prepared by W/O/W double emulsions solvent evaporation method was determined by employing L9(3⁴)orthogonal test design,including the effect of PLGA concentration,polyvinyl alcohol(PVA) concentration,oil/water ratio,and choice of stabilizers and quantity used.In the sustained-release study of IFNαmicrospheres,the in vitro release behaviors of IFNαmicrospheres were studied firstly by an improved release method.We compared the in vitro release profiles of IFNαmicrospheres,which prepared by five different viscosities, 0.17dL/g,0.39dL/g,0.60dL/g,0.89dL/g,1.13dL/g and five different concentrations,5%,10%,15%,20%,25%.The optimum formulation of IFNαmicrospheres preparation was also further perfected by the results of the in vitro release.In vitro release profile of IFNαmicrospheres prepared by 15%0.89dL/g PLGA met the criteria of sustained release formulation preferably and it was able to prolong drug action.Higuchi equation was Q=2.203t^1/2+25.605,r=0.9023.The accumulated amount of IFNαreleased was 75%in 15 days.Moreover,the biological activity of IFNαwas measured by Cytopathic Inhibition Assay suggesting that bioactive IFNαhad been firmly released from microsphere for more than 7 days.The pharmacokinetic experiments of this microspheres showed an obvious sustained fashion,and the relative bioavailability of IFNαmicrospheres to IFNαwas about 200%.The IFNαmicrospheres prepared by optimum formulation were white powder with fine fluidity.The surface morphology of microspheres had a spherical shape with a smooth and porous surface.Particle size of IFNαmicrospheres were nearly normality and the average diameter of microspheres was 65.84μm.The bulk density and the angel of repose of microspheres were 0.1856g/ml and 29.42°respectively.The moisture absorption of microspheres was low for their hygroscopic ratio less than 1%under the condition of 90% relative humidity.However,IFNαmicrospheres should be stored in airproof and dry container since humidity could accelerate IFNαdegradation.The DSC curve of microspheres confirmed that IFNαwas loaded within the microspheres,rather than adsorbed on their surfaces.For dichloromethane was used to prepare microspheres,the residual quantity of dichloromethane in microspheres should be measured.The amount determined by gas chromatography and mass spectra was lower than the criteria of Chinese Pharmacopeia.The biodegradability of PLGA microspheres was investigated by an improved method in vitro and by intramuscular injection in rats in vivo.The biodegradable speeds of IFNαmicrospheres in vivo were faster than those in vitro.After six weeks,the degradable percentage of IFNαmicrospheres was 80%in vitro and 100%in vivo,so that IFNαmicrospheres would not be accumulated in body.Biocompatibility of microspheres had been observed for six weeks by intramuscular injection in rats.It showed that IFNαmicrospheres were biocompatible for only slight inflammatory response but no pathological change at the site of injection.The results of the above study provided a useful exploration for the experimental methods and theories of peptide and protein microspheres and a scientific basis for developing of IFNαmicrospheres as a new drug to cure hepatitis.