microencapsulation is a useful method for prolonging drug release from dosage form and reducing adverse effect . recently , dosage forms that can precisely control the release rates and target drugs specific body site have made an enormous impact in the formulation and development of novel drug delivery system . microspheres are one of the multiparticulate delivery systems and are prepared to obtain prolonged or controlled drug delivery , to improve bioavailability or stability and to target drug to specific sites . microspheres can also offer advantages like limiting fluctuation within therapeutic range , reducing side effects , decreasing dosing frequency , and improving patient compliance [ 5 , 6 ] . ethylcellulose ( ec ) that is a hydrophobic and ph - independent polymer has been widely used in the prepared sustained release dosage forms of a water - soluble material [ 710 ] . the substance encapsulated in the microsphere is released under the influence of a specific stimulus at a specified stage . whereas interaction between dissolution media , polymer , and drug is the primary factors in release control [ 12 , 13 ] , various formulation variables influence drug release rate to greater or lesser extent . thus , drug loading [ 14 , 15 ] , drug / polymer ratio [ 1618 ] , and drug particle size   have been shown to affect drug release from ec matrices . stavudine is a nucleotide reverse transcriptase inhibitors and primarily used in the treatment of one of the most common chronic disease of the planet , aids . it has short biological half - life 0.81.5  h , and low daily dose of 30  mg is required [ 20 , 21].the frequency of dosing is more . to overcome this problem response surface methodology ( rsm ) is widely practiced approach in the development and optimization of drug delivery devices . based on the principle of design of experiments ( does ) , the methodology encompasses the use of various types of experimental designs , generation of polynomial equations , and mapping of the response over the experimental domain to determine the optimum formulation(s ) [ 2227 ] . the technique requires minimum experimentation and time , thus proving to be far more effective and cost - effective than the conventional methods of formulating dosage forms . the current study aims at developing and optimizing microspheres of stavudine using rsm , as it may prove to be more productive than the conventional systems by virtue of prolongation of drug residence time in gastrointestinal tract . further , microsphere of the drug would involve relatively more economical and less complicated technology vis -  - vis many other drug delivery devices . computer - aided optimization technique , using a full factorial design , was employed to investigate the effect of 2 independent variables ( factors ) ( i.e. , the drug - to - polymer ratio and stirring speed ) on particle size , encapsulation efficiency , and drug release . acetone and light liquid paraffin were obtained from ranbaxy fine chemical ltd . , new delhi , india used as dispersion media . microspheres of ethylcellulose were prepared by emulsion solvent diffusion technique , using ec as a polymeric retardant material . polymer was dissolved in 5  ml organic solvent consisting of acetonitrile and dichloromethane ( 1  :  1 ratio ) . the resultant solution was extruded through a syringe ( g 20 ) into the solution of drug in aqueous medium ( 2  ml ) under stirring at 500  rpm using mechanical stirrer ( remi motors , india ) for 5 minutes , to form primary emulsion ( w / o ) . the w / o primary emulsion was slowly added to 50  ml of light liquid paraffin containing 0.5% span-80 , 1% ethylcellulose ( 50  mg ) , 1%  w / v magnesium stearate ( 50  mg ) as a tensioactive agent , saturation , and droplet stabilizer in the processing medium , respectively , under stirring in different rpm to form w / o / o multiple emulsion.the whole system was stirred for about 3  h. after stirring process was over , the light liquid paraffin was decanted off and microspheres formed were collected by filtration using ordinary filter paper ( pore size 25  m ) and treated with petroleum ether ( 4060c ) for several times to completely remove the oil . microspheres then air dried at room temperature for 12  h and collected for further studies . the drug - to - polymer ratio and stirring speed were varied in batches f1 to f9 . stavudine was estimated by ultraviolet visible ( uv / vis ) spectrophotometric method ( hitachi , u-1700 , japan ) . aqueous solution of stavudine was prepared in phosphate buffer ( ph 6.8 ) , and absorbance was measured on uv / vis spectrophotometer at 266  nm . the method obeys beer 's law in the concentration range of 5 to 50  g / ml . when a standard drug solution was analyzed repeatedly ( n = 6 ) , the mean error ( accuracy ) and relative standard deviation ( precision ) were found to be 0.7% and 1.2% , respectively . microspheres ( 50  mg ) were crushed in a glass mortar and pastle , and the powdered microsphere were suspended in 50  ml phosphate buffer ( ph 6.8 ) . the resulting mixture was shaken by the magnetic stirrer for 24  h. the solution was filtered , and the filtrate was analyzed for the drug content . the drug entrapment efficiency was calculated using the following formula :  
 ( 1)drug  entrapment  efficiency  = practical  drug  contenttheoritical  drug  content100 . particle size of the microspheres was measured by laser light scattering technique ( mastersizer 2000 , malvern , uk ) . the sizes of the completely dried microspheres of different formulations were measured by dry sample technique using dry sample adapter . the completely dried particles were placed on the sample tray with an in - built vacuum , and compressed air system was used to suspend the particles . after measurement of particle size of each sample , the dry sample adopter was cleaned thoroughly to avoid cross contamination . each batch was analyzed in triplicate , but the average values were considered in data analysis . 
 in vitro drug release study of microspheres were carried out using usp xxiv paddle type apparatus ( campbell electronic , mumbai , india ) at 37  1c and at 100  rpm using 900  ml phosphate buffer ( ph 6.8 ) . microsphere equivalent to 50  mg of stavudine were used for the test . at predetermined intervals , 5  ml of aliquots were withdrawn and replaced by the same volume of fresh media . aliquots were filtered through a 0.45  m membrane filter , diluted suitably , and analysed spectrophotometrically . percent of drug dissolve at different time intervals was calculated using lambert - beer 's equation ( y = 0.0463x  0.006 ) describe above . the samples were then filtered through membrane filter ( 0.45  m ) and diluted suitably . the amount of drug present in the solution was then analyzed spectrophotometrically at 225  nm using uv - visible spectrophotometer ( shimadzu , uv-1700 , japan ) . the surface topography of the prepared microspheres was examined by scanning electron microscope ( hitachi , s-3600n , japan ) . the samples were fixed on brass stub using double - sided tape and then gold - coated in vacuum by a sputter coater . a statistical model incorporating interactive and polynomial terms was used to evaluate the responses : 
 ( 2)y = b0+b1x1+b2x2+b12x1  x2+b11x12+b22x22 , 
 where y is the dependent variable , b0 is the arithmetic mean response of the 9  runs . the main effects ( x1 and x2 ) represent the average result of changing one factor at a time from its low to high value . the interaction terms ( x1x2 ) show how the response changes when 2 factors are simultaneously changed . the polynomial terms ( x1  and  x2 ) are included to investigate nonlinearity . microspheres of stavudine were prepared by emulsion solvent diffusion technique using ec as a polymer due to its hydrophobicity and release - controlling properties . at first in trial batch , viscosity of the polymer solution is optimized since it is an important factor related to microspheres as reported by lee et al . . polymer concentration of 0.5% , 1% , and 2% w / v were selected for preliminary trials . flake formation was observed when ethylcellulose concentration was used at a level of 0.5% , whereas maximum sphericity was observed at the 1% level . the ethylcellulose solution was found to be too viscous to pass through the syringe when used at the 2% level . therefore , 1% was found to be the optimum concentration for the entire factorial batch . the volume of secondary oil phase is an important factor as related to the formulation of microspheres . the volume of light liquid paraffin to be considered for the factorial batch was selected on the basis of in vitro release study . different volume of light liquid paraffin ( from 20 to 60  ml ) was used in trial batch . as the volume of light liquid paraffin is increased from 20  ml to 50  ml , the in vitro release of stavudine is significantly ( p < .05 ) decreased . on the other hand , insignificant ( p > .05 ) decrease of the in vitro drug release was observed when the light liquid paraffin volume was increased from 50  ml to 60  ml . concentration of span-80 plays an important role in the formulation of microspheres prepared by emulsion solvent diffusion technique . therefore , suitable concentration of span-80 for factorial batch was selected by taking into account their aggregation phenomenon . span-80 in the concentration of 0.5% was found suitable to prevent aggregation of the microspheres .   the sem photograph ( figure 1 ) revealed that the drug - loaded microspheres are spherical . microspheres prepared containing higher amount of the polymer ( 1  :  3 drug  :  polymer ratio ) exhibited smoother surfaces than those prepared taking a lower amount of the polymer ( 1  :  1 and 1  :  2 ) . irregular surfaces and larger sizes of the microspheres were observed for those prepared with a lower amount of the polymer . as the drug - to - polymer ratio was increased , more spherical microspheres with smooth surfaces were obtained as suggested earlier .   on the basis of the preliminary trials a 3 full factorial design was employed to study the effect of independent variables ( i.e. , drug - to - polymer ratio [ x1 ] and the stirring speed [ x2 ] ) on dependent variables ( mean particle size , drug entrapment efficiency , and t80% ) . the results depicted in table 1 clearly indicate that all the dependent variables are strongly dependent on the selected independent variables as they show a wide variation among the 9 batches ( b1 to b9 ) . the fitted equations ( full models ) relating the response ( i.e. , mean particle size , drug entrapment efficiency , and t80% ) to the transformed factor the polynomial equations can be used to draw conclusions after considering the magnitude of coefficient and the mathematical sign it carries ( i.e. , positive or negative ) . the high values or correlation coefficient ( table 2 ) for the dependent variables indicate a good fit . mathematical relationship generated using multiple linear regression analysis for the studied variables are expressed as follows : 
 ( 3)mean  particle  size  = 244.38 + 32.83x18.83x21.50x1x2  + 1.67x120.33x22 , 
 ( 4)entrapment  efficiency  = 59.07 + 8.33x13.50x2 + 0.50x1x2  1.24x12 + 1.26x22 , 
 ( 5)t80%=348.79 + 124.33x119.67x224.00x1x2  + 79.72x1242.28x22 . all the polynomial equations were found to be statistically significant ( p < 0.01 ) , as determined using anova ( table 3 ) , as per the provision of design expert software . particle size analysis of microspheres was found to be in the range of 206290  m ( table 1 ) . results of the equation indicate that the effect of x1 ( drug - to - polymer ratio ) is more significant than x2 ( stirring speed ) . moreover , stirring speed had a negative effect on the particle size ( i.e. , as the stirring speed increased , the particle size decreased ) . as the stirring speed was increased , the size of microdroplet of the emulsion was decreased resulting in the formation of smaller size microparticles . these findings are similar to those reported previously ( 27,28 ) . figures 2(a ) and 2(b ) depict a linear trend of mean particle size in an ascending order with an increase in each variable . it also shows that drug - to - polymer ratio has a comparatively greater influence on the response variables than stirring speed . the drug entrapment efficiency is important variable for assessing the drug loading capacity of microspheres . this parameter is dependent on the process of preparation , physicochemical properties of drug , and formulation variables . the drug entrapment efficiency varied from 47% to 70% and showed good correlation coefficient ( 0.9970 ) . result of equation indicates the effect of x1 ( drug - to - polymer ratio ) is more significant than x2 ( stirring speed ) . moreover , stirring speed had a negative effect on drug entrapment efficiency ( i.e. , the stirring speed increased , the particle size decreased , and thus drug entrapment efficiency decreased ) . as the ratio of drug - to - polymer increased , encapsulation efficiency increased ; this is due to the fact that higher ratio of drug - to - polymer would produce large size droplets with decreased surface area , such that diffusion of drug from such microsphere will be slow , resulting in higher encapsulation efficiency . figures 3(a ) and 3(b ) also exhibit that entrapment efficiency vary in a nonlinear manner , but in ascending pattern with an increase in each variable . . however , the effect of drug - to - polymer seems to be more pronounced as compared to stirring speed . the release profiles of formulations appear to be slow release with negligible burst effect . the formulations with lower levels of drug - to - polymer ratio exhibited higher initial burst in drug release . this could be attributed to the dissolution of drug present initially at the surface of the microspheres . however , the formulations showed little burst effect at higher drug - to - polymer ratio , ratifying better sustenance of drug released . the values of t80% enhanced markedly from 264 minutes , observed a low levels of drug - to - polymer ratio and stirring speed , to as high as 571 minutes , observed at high level of drug - to - polymer ratio and stirring speed . batch f7 exhibited a high t80% of 517 minutes and since it is a promising candidate for achieving drug release up to 12  hrs . the percentage in vitro drug release is highly dependent on drug - to - polymer ratio and stirring speed . results depicted in table 2 indicate that the effect of drug - to - polymer ratio ( x1 ) is more significant than stirring speed ( x2 ) . the stirring speed had a negative effect on t80% because as the stirring speed increased , the particle size of microspheres is increased , resulting in decrease of drug release . on the other hand , as the drug - to - polymer ratio is increased , the drug loading was decreased , resulting in decrease of drug release from microspheres . on the other way , increase in polymer matrix into the microspheres leads to an increased diffusional path length and thereby decreased the overall drug release from microspheres . furthermore , smaller microspheres are formed at lower polymer concentration and have larger surface area exposed to dissolution medium . figures 4(a ) and 4(b ) show that t80% vary in nonlinear fashion , but in ascending pattern with an increase of each variable . the contour plot ( figure 5(b ) ) shows that drug - to - polymer ratio has a comparatively greater influence on the response variable than stirring speed . the optimum formulation was selected based on the criteria of attaining complete and controlled release with highest possible entrapment efficiency . upon the following maximizing criteria were adopted : mean particle size < 300  m , entrapment efficiency > 60% , and t80% > 540 minutes . accordingly , formulation f7 was ranked as best batch . in order to determine the mechanism of drug release from the formulation f7 , the data obtained in vitro release study were fitted to the korsemeyer - peppas model in order to determine the n  value of all the formulations was between 0.5 and 1 indicating that the mechanism of drug release was non - fickian type diffusion . sustained drug release in the current study indicates that the hydrophobic matrix microspheres of stavudine , prepared using ec , can successfully be prepared by emulsion solvent diffusion technique . the results of a 3 factorial design revealed that the drug - to - polymer ratio and stirring speed are imperative to acquire sustained release and entrapment efficiency . the microspheres of best batch exhibited mean particle size of 290  m and entrapment efficiency of 70% . the t80% of 571 minutes indicates that the microspheres of stavudine could sustain the release of the drug for more than 12  h.