the systems for controlled delivery 
 of the medicaments in the body are causing real revolution in the medicine and 
 pharmacy in the recent years , and all in favor of better medical treatments of 
 the patients . using the system for the controlled and balanced release of 
 medicaments , opposing to standard and conventional methods , constant and uniform concentration of medicament in the body is achieved throughout longer period of time .
copolymer poly(d , l - lactide - co - glycolide ) is used for the 
 controlled delivery of several classes of medicaments like anticancer agents , 
 anti - hypertensive agents , immunomodulatory drugs , hormones , and macromolecules 
 like nucleic acid , proteins , peptides , antibodies , dlplg nanospheres are very 
 efficient mean of transdermal transport of medicaments in the body , for 
 example , ascorbic acid .
dlplg 
 polymer particles allow the encapsulation of the medicament within the polymer 
 matrix , where the principle requirement for the controlled and balanced release 
 of the medicament in the body is the particle 's ideal spherical shape and narrow distribution of its size .
the size and 
 shape of the particles play key role in their adhesion and interaction with the 
 cell .
dynamic of the release ( pace and concentration ) depends of the 
 morphology , that is , structure of the copolymer .
the chemical structures , 
 molecular weight , composition , as well as the synthesis conditions , are 
 parameters which influence the final morphology of the polymer . the direct 
 relation between these parameters and morphology is inadequately examined thus 
 making it a topic of many researches . depending on the nature and matrix of the selected 
 material , methods of obtaining polymer particles can be divided in general into 
 dispersion of the polymer solution method , polymerization of the monomer method , 
 and coacervation [ 36 ] .
the plga spheres obtained with emulsion process are in 
 range of 150200 m , 45 m , 30 m . with 
 modified emulsion method ,
further modification of the process for synthesis of the particles , that is , emulsification solvent evaporation method , the obtained particles are in nanometer scale of 570970 nm   and 244260 nm [ 1214 ] .
the latest researches in this field indicated the possibility of producing dlplg spheres with average diameter 
 under 100 nm .
controlling the conditions of 
 obtaining dlplg by solvent / nonsolvent method , changing the parameters like 
 aging time , after adding nonsolvent , time and velocity of centrifugal 
 processing , it is possible to influence on morphology ( size and shape ) and 
 uniformity of dlplg polymer powder .
dlplg powder with short aging time with nonsolvent and longest time and velocity of the centrifugal processing has 
 smallest particles and highest uniformity .
dlplg 
 copolymer has potential to be used for transport of ascorbic acid in the body , 
 thus considerably increasing its efficiency .
ascorbic acid reduces free radicals , and in that way damages created by oxidative stress which is a root cause of , or at least 
 associated with , many diseases are minimized .
the aim of this research is obtaining the 
 nanoparticles of copolymer poly(d , l - lactide - co - glycolide ) in which ascorbic 
 acid is encapsulated , as well as examining the influence of the synthesis 
 method on morphological characteristics of poly(d , l - lactide - co - glycolide ) 
 particles with the different content of ascorbic acid .
poly(d , l - lactide - co - glycolide ) 
 ( dlplg ) was obtained from durect , lactel , adsorbable 
 polymers international and 
 had a lactide to glycolide ratio of 50 : 50 .
time of complete resorption of 
 this polymer is 48 weeks . 
 molecular weight of ascorbic acid was 176.13 g / mol .
copolymer powder dlplg was obtained 
 by means of physical methods from commercial granules using solvent / nonsolvent 
 systems ( figure 1 ) . 

commercial granules poly(d , l - lactide - co - glycolide ) ( 0.05 g ) 
 were dissolved in 1.5 ml of acetone and , after approximately two hours , 2 ml of 
 methanol was added into solvent mixture .
the polymeric solution thus obtained 
 was very slowly poured into 20 ml of aqueous pva solution ( 0.02% w / w ) while 
 continuous stirring at 1200 rpm by a stirrer .
pva is used 
 as a stabilizer which creates negative charge of the dlplg particles , that is , 
 it creates negative zeta potential . by creating specific zeta potential
the ascorbic acid was encapsulated into the 
 polymer matrix by means of homogenization of water and organic phases .
the 
 water solution with the variable ratio of the ascorbic acid was added to the polymer 
 solution .
this was followed by the precipitation using alcohol methanol . in the 
 particles of dlplg copolymer , different concentration of ascorbic acid
has been 
 encapsulated with ratios 85% dlplg to 15% ascorbic acid , 70% dlplg to 30% 
 ascorbic acid , 50% dlplg to 50% ascorbic acid , and 30% dlplg to 70% ascorbic acid .
the ir measurements were performed on 
 perkin - elmer 983 g infrared spectrophotometer , using the kbr pellet technique , 
 in the frequency interval of 4004000 cm .
the morphology of obtained particles 
 of dlplg was examined by scanning electron microscope ( sem ) jeol jsm-646olv . 

the powder samples for sem analysis were coated with gold using the physical 
 vapor deposition ( pvd ) process .
samples were covered with gold ( scd 005 sputter 
 coater ) , using 30 ma current from the distance of 50 mm during 180 seconds . the particle size and morphology
were 
 examined using the area analysis method [ 18 , 19 ] by semiautomatic image analyzer ( videoplan , kontron ) , connected with a scanning electron microscope ( sem ) . from 200 to 300 particles in the sem were measured and the following 
 parameters were determined : area section aa , perimeter lp , maximal diameter of 
 the particle dmax , feret x and feret y , and form factor ( fl ) ( figure 2 ) .
release of the ascorbic acid from dlplg particles in vitro in physiological solution ( 0.9% sodium chloride in water ) was studied with uv spectroscopy .
the uv measurements were performed on 
 perkin - elmer lambda 35 uv - v is spectrophotometer in the frequency interval of 
 200400 nm .
the ir spectra in figure 3 illustrate 
 all characteristic groups for copolymer poly(d , l - lactide - co - glycolide ) .
the ir 
 spectra of dlplg show peaks at 2994 , 2946 , 2840 ( ch bend ) , 1769 ( c = o 
 ester ) , 1460 , 1424 , 1371 ( ch3 ) , 1150 , 
 1069 984 ( co stretch ) , 732 509 ( ch - bend ) cm while 
 the band on 31003600 cm belongs 
 to the oh group of the water molecule .
comparing the obtained ir spectra for 
 dlplg and ascorbic acid ( figure 4 ) with the ir spectra charecteristics for 
 ascorbic acid shown in the literature [ 21 , 22 ] , it is confirmed that obtained nanoparticles are composed of 
 poly(d , l - lactide - co - glycolide ) and ascorbic acid . besides the characteristic groups for copolymer dlplg ,
the four oh bands of ascorbic acid could be assigned by means 
 of infrared investigations at 3528 , 3411 , 3317 , 3217 cm . 

the spectra show bands that can be assigned to ch3 , ch2 , 
 or ch groups in the ascorbic acid environment at 2720 cm and the 
 spectra also clearly show the band corresponding to c = o groups at 2916 cm .
the bands that correspond to the wave number 1754 cm belong to c = c groups , 1673 cm coc , 
 and 1020 cm co , 
 respectively .   the morphological characteristics of 
 the obtained dlplg particles , with and without encapsulated ascorbic acid , were 
 examined with a scanning electron microscope . from the sem recordings of dlplg 
 particles without ascorbic acid ( figure 5(a ) ) , it is visible that the 
 particles have spherical shape , smooth surface , low level of agglomeration , and 
 high level of uniformity  higher than other samples . from the sem recordings 
 of the second sample ( figure 5(b ) ) , where dlplg copolymer has encapsulated ascorbic acid in ratio dlplg / ascorbic acid 85/15% , it is visible that 
 particles also have spherical shapes , that is , spherical shape of the initial 
 dlplg
dlplg / ascorbic acid 85/15% nanoparticles are 
 uniform with sizes from 130 to 200 nm depending on which stereological 
 parameters are considered ( dmax , maximum diameters , feret x , or feret y ) .
the 
 particles of the sample dlplg / ascorbic acid 70/30% ( figure 5(c ) ) also have spherical shapes , but their sizes are increased . in case of the fourth sample , 
 dlplg / ascorbic acid 50/50% ( figure 5(d ) ) uniformity is perturbated , particles have both spherical and irregular shapes and they are much agglomerated . for 
 the fifth sample , dlplg / ascorbic acid 30/70% ( figure 5(e ) ) ,
the stereological analysis is giving us 
 the parameters which are characterizing the particle sizes ( area section 
dmax , and feret 's diameters ) and parameter which is characterizing 
 the particle shape ( perimeter form factor 
maximum , and mean values were recorded and presented in table 1 . based on the obtained results of the 
 stereological analysis of dlplg particles , it is visible that they are uniform , 
 their average mean size varies from 0.15 to 0.23 m depending on the 
 stereological parameter taken in consideration ( dmax , feret x , or feret y ) 
 ( table 1 ) . 

dmax values range from 0.09 to 0.39 m with particle 's mean size 
 0.23 m ( figure 6 ) .
figures 
  7 and 8 present comparative results of dlplg particles with and without ascorbic acid based on their area section and 
 perimeter form factor . from the comparative results of the 
 stereological analysis of the area section ( aa ) of dlplg with and
without 
 encapsulated ascorbic acid ( figure 7 ) as well as comparative results of the 
 perimeter form factor ( fl ) , ( figure 8) we can see that dlplg particles without 
 ascorbic acid have the smallest area section ( minimum value for aa is 0.02 mand maximum is 0.08 m ) and the highest mean value of perimeter 
 form factor which is 0.89 . nanoparticles dlplg / ascorbic acid 85/15% have 
 minimum dmax of 0.09 m and maximum dmax of 0.49 m , 
  where their mean size is 0.20 m 
 ( figure 6 ) .
the mean value of the area section is 0.03 m ( figure 7 ) and of the perimeter form factor is 0.87 ( figure 8) . for particles dlplg / ascorbic acid 70/30%
, minimum dmax is 0.30 m 
 and maximum dmax is 2.59 m , where their mean size is 0.67 m ( figure 6 ) , which indicates that the uniformity 
 is decreased and size is increased .
the mean value of the area section is 0.41 m ( figure 7 ) and of the perimeter 
 form factor is 0.77 ( figure 8) . for particles
dlplg / ascorbic acid 50/50% , minimum dmax is 0.28 m 
 and maximum dmax is 4.51 m , where their mean size is 1.60 m ( figure 6 ) .
the 
 mean size of the area section is 2.30 m ( figure 7 ) and of the perimeter form factor is 0.74 ( figure 8) . in case of dlplg / ascorbic acid 
 30/70%
the release amount of the ascorbic acid 
 from the polymer particles was determined periodically during the eight weeks 
 with uv spectroscopy .
a calibration curve of the ascorbic acid in physiological 
 solution at different concentrations has been prepared using the specific 
 absorbance peak of the ascorbic acid at 264 nm . 
 
figure 9 shows the dependence of the maximum absorption from the degradation time in cases of dlplg without ascorbic 
 acid , dlplg / ascorbic acid 85/15% , dlplg / ascorbic acid 70/30% , and 
 dlplg / ascorbic acid 50/50% .
this absorbance is correlated with the calibration 
 curve and amount of ascorbic acid is determined in percentages .
figure 10 gives cumulative curves of the release of the ascorbic acid in percentages over the period of 
 time of the degradation .
figure 10 also shows the relative review in 
 percentages of the ascorbic acid release in periods of up to two days , 211 , 1117 , 1724 , 2431 , 
 3139 , 3946 , and 4655 
 days . in the first 24 days of the degradation , for all samples ,
less than 10% 
 of the encapsulated ascorbic acid have been released . 
  for all dlplg / ascorbic 
 acid samples ,
the overall quantities of the encapsulated ascorbic acid have 
 been released in 8 weeks of the degradation .
the particles obtained with solvent / nonsolvent physical 
 method and technique of the centrifugal processing have potential use in transdermal systems for controlled delivery of ascorbic acid .
it is possible to encapsulate ascorbic acid into dlplg particles in various concentrations thus 
 producing particles with different morphological characteristics .
the 
 nanoparticles of dlplg / ascorbic acid with lesser ratio of ascorbic acid have 
 higher uniformity , lower level of agglomeration , and smaller sizes .
the 
 nanoparticles of dlplg / ascorbic acid 85/15% have spherical shapes and their 
 sizes are from 130 to 200 nm .
 this paper is covering new , simplistic method of obtaining the system for controlled delivery of the ascorbic 
 acid . 
 copolymer poly ( d , l - lactide - co - glycolide ) ( dlplg ) nanoparticles are produced using physical method 
 with solvent / nonsolvent systems where obtained solutions were centrifuged . 
 the encapsulation of the 
 ascorbic acid in the polymer matrix is performed by homogenization of water and organic phases . 
 particles of 
 the dlplg with the different content of ascorbic acid have different morphological characteristics , that is , variable degree of uniformity , agglomeration , sizes , and spherical shaping . 
 mean sizes of nanoparticles , which contain dlplg / ascorbic acid in the ratio 85/150% , were between 130 to 200 nm depending on which stereological parameters are considered ( maximal diameters dmax , feret x , or feret y ) . by introducing up to 15% of ascorbic acid , the spherical shape , size , and uniformity of dlplg particles are preserved . 
 the samples were characterized by infrared spectroscopy , scanning electron microscopy , stereological analysis , and ultraviolet spectroscopy . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS AND DISCUSSION
4. CONCLUSIONS