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Colloids and Surfaces B: Biointerfaces 59 (2007) 215–223

Fabrication, in vitro degradation and the release behaviours ofpoly(dl-lactide-co-glycolide) nanospheres containing ascorbic acid

Magdalena Stevanovic a, Jasmina Savic b, Branka Jordovic c, Dragan Uskokovic a,∗a Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade 11000, Serbia

b Laboratory of Physical Chemistry, Vinca Institute of Nuclear Sciences, Belgrade 11000, Serbiac Faculty of Technical Sciences, Cacak 32000, Serbia

Received 13 February 2007; received in revised form 9 May 2007; accepted 16 May 2007Available online 23 May 2007

bstract

Ascorbic acid (vitamin C) is essential for preserving optimal health and is used by the body for many purposes. The problem is that ascorbic acidasily decomposes into biologically inactive compounds making its use very limited in the field of pharmaceuticals, dermatological and cosmetics.y encapsulating the ascorbic acid into a polymer matrix it is assumed that its chemical instability can be overcome as well as higher, more efficientnd equally distributed concentration throughout extended period of time can be achieved. This paper is describing the process of obtaining poly(dl-actide-co-glycolide) (DLPLG) nanospheres (110–170 nm) using chemical method with solvent/non-solvent systems where obtained solutionsave been centrifuged. The encapsulation of the ascorbic acid in the polymer matrix is performed by homogenisation of water and organic phases.

anoparticles of the copolymer DLPLG with the different contents of the ascorbic acid have different morphological characteristics, i.e. variableegree of uniformity, agglomeration, sizes and spherical shaping. The degradation of the nanospheres of DLPLG, DLPLG/ascorbic acid nanopar-icles and release rate of the ascorbic acid were studied for 8 weeks in a physiological solution (0.9% sodium chloride in water). The samples haveeen characterised by infrared spectroscopy (IR), scanning electron microscopy (SEM), stereological analysis and ultraviolet (UV) spectroscopy. 2007 Elsevier B.V. All rights reserved.

ticles;

ctorbgssebtbn

eywords: Nanospheres; Poly (dl-lactide-co-glycolide)/ascorbic acid nanopar

. Introduction

Poly (esters) based on polylactide (PLA), polyglycolidePGA), polycaprolactone (PCL), and their copolymers haveeen extensively employed as systems for drug delivery1–6]. Degradation of these materials yields the correspond-ng hydroxyl acids, making them safe for in vivo use. DLPLGegrades by hydrolysis of its linkages in the presence of water.scorbic acid is water-soluble vitamin, which cannot be syn-

hesised and stored in the body. It has variety of biological,harmaceutical and dermatological functions. Vitamin C pro-otes collagen biosynthesis, provides photoprotection, causeselanin reduction, scavenges free radical, and enhances the

mmunity (anti-virus effect), etc. [7]. Nevertheless, ascorbic acids very unstable to air, light, heat, moisture, metal ions, oxygen,nd base, and it easily decomposes into biologically inactive

∗ Corresponding author.E-mail address: uskok@itn.sanu.ac.yu (D. Uskokovic).

tcroeds

927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2007.05.011

Degradation; Drug delivery; Drug release

ompounds such as 2,3-diketo-l-gulonic acid, oxalic acid, l-hreonic acid, l-xylonic acid and l-lyxonic acid [8]. In order tovercome the chemical instability of the ascorbic acid numerousesearches have been staged toward its encapsulation or immo-ilisation [9–12]. The ascorbic acid introduced in the body in thereater portion is isolated from the body. However, the encap-ulated ascorbic acid within the polymeric matrix should haveignificantly higher efficiency. DLPLG nanospheres are veryfficient means of transdermal transport of medicaments in theody, e.g. ascorbic acid [13]. One of the basic requirements forhe controlled and balanced release of the medicament in theody is its ideal spherical shape of the DLPLG particles andarrow distribution of their sizes. The size and shape of the par-icles play key role in their adhesion and interaction with theell. Polymer degradation, also, plays a key role in medicamentelease from sustained release polyester systems, therefore in

rder to elucidate the mechanism governing release, it appearsssential to analyse the in vitro degradation behaviour of theseevices [14–18]. Depending on the nature and matrix of theelected material, the methods of obtaining polymer particles

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an be divided in general to dispersion of the polymer solutionethod, polymerisation of the monomer method and coacer-

ation [19–21]. The PLGA spheres obtained with emulsionrocess are in the range of 150–200 �m [22], 45 �m [23],0 �m [24]. With modified emulsion method, the particle sizesre decreased to 10 �m [25]. Further modification of the pro-ess for synthesis of the particles i.e. emulsification solventvaporation method, the obtained particles are in nanometercale of 570–970 nm [26] and 240–260 nm [27–30]. The latestesearches in this field indicated the possibility of producingLPLG spheres with average diameter under 100 nm [31].he spherical shaping and the particle sizes are being greatly

nfluenced by the molecular weight of the polymer, solvent/non-olvent ratio, aging time with non-solvent, type of the stabilisersed, stirring velocity, centrifugation force, etc. [2,32–35]. Con-rolling the conditions of obtaining DLPLG by chemical methodolvent/non-solvent, changing the parameters like aging timeafter adding non-solvent), time and velocity of centrifugal pro-essing; it is possible to influence the morphology (size andhape) and uniformity of DLPLG polymer powder [32]. DLPLGowder with the shortest aging time with non-solvent and theongest time and velocity of the centrifugal processing has themallest particles and the highest uniformity [32].

The aim of this research is obtaining poly(dl-lactide-co-lycolide) nanospheres, the encapsulation of the ascorbic acidnto the DLPLG particles and the examining of the degradationf the DLPLG particles without and with encapsulated ascorbiccid in vitro conditions. The effect of degradation on the mor-hology of the DLPLG nanospheres and DLPLG/ascorbic acidanoparticles has also been examined. In this study, the relation-hip between polymer matrix degradation and the ascorbic acidelease profile is drawn.

. Experimental procedures

.1. Materials

Poly(dl-lactide-co-glycolide) (DLPLG) was obtained fromurect, Lactel, Adsorbable Polymers International and had a

actide to glycolide ratio of 1:1. Molecular weight of polymeras 40,000–50,000 g/mol. Molecular weight of ascorbic acid

s 176.13 g/mol. Polyvinyl pyrrolidone (povidone, PVP) wasbtained from Merck Chemicals Ltd. (k-25, Merck, Germany).he degradation medium used was physiological solution (0.9%odium chloride in water) of pH 7 at 20 ◦C. All other chemicalsnd solvents were of reagent grade.

.2. Methods

.2.1. Preparation of poly(DL-lactide-co-glycolide)anospheres

The DLPLG nanospheres have been prepared with chemicalethods from commercial granules using solvent/non-solvent

ystems. Commercial granules of poly(dl-lactide-co-glycolide)ave been dissolved in the acetone and, after approximately 2 h,ethanol has been added into the solvent mixture. DLPLG pre-

ipitated by the addition of methanol and the solution became

obss

B: Biointerfaces 59 (2007) 215–223

hitish. The polymeric solution thus obtained has been verylowly poured into 20 ml of aqueous PVP solution (0.05%, w/w)hile continuously stirring at 1200 rpm by a stirrer. After that

he solution has been centrifuged (Centrica, Ependorf), decantednd dried. Time and velocity of the centrifugal processing haseen 120 min on 4000 rpm. Using PVP as the stabiliser, neg-tively charged DLPLG particles are produced, i.e. specificeta potential is produced, which prevents their agglomeration36–39]. By stirring on the magnetic stirrer under influence of thengular velocity and fricative frequency, the particles are formed,here those particles’ shapes and dimensions are finalised under

nfluence of the centrifugation force.

.2.2. Encapsulation of the ascorbic acid in DLPLGanospheres and yielding in preparation

The ascorbic acid has been encapsulated into the polymeratrix by means of homogenisation of the water and organic

hases. Fifty milligrams of DLPLG commercial granules haveeen dissolved in the 1.5 ml of acetone. The DLPLG has beenissolving in acetone approximately 2 h at room temperature.fterwards, the aqueous solution of the ascorbic acid has been

dded in DLPLG solution in acetone while continuously beenomogenised at 200 rpm during 30 min. Concentration of thescorbic acid in the water has been varied in order to obtainedanoparticles with different ratio of DLPLG and ascorbic acidDLPLG/ascorbic acid 85/15%, DLPLG/ascorbic acid 70/30%nd DLPLG/ascorbic acid 50/50%). Accordingly, the weightsf the ascorbic acid in 2 ml of water were 8.80 mg, 21.42 mgnd 50.00 mg. This has been followed by the precipitation usingml of methanol. Thus, obtained solution has been very slowlyoured into 20 ml of aqueous PVP solution (0.05%, w/w) whileontinuously stirring at 1200 rpm. After that solution has beenentrifuged at 4000 rpm for 120 min. The supernatant has beenecanted and stored for analysis of the ascorbic acid concentra-ion by spectrophotometry. Particles, dried at room temperature,ave been weighed and the yield was calculated as a percentage40] using equation:

ield =[

(weight of particles)

(weight of polymer + weight of the ascorbic acid)

]

×100

.2.3. Loading amount and loading efficiencyThe amount of the ascorbic acid encapsulated during

LPLG/ascorbic acid nanoparticles synthesis process was deter-ined as follows (a method described by Liu et al. [41]). The

scorbic acid absorbs light with a wavelength of 264 nm. Basedn the measuring of the absorbance at 264 nm of the solutionith known concentration of the ascorbic acid a calibration curveas prepared. The liner relationship between light absorbance

t 264 nm and the ascorbic acid is shown according to theeer’s Law A = εbc (where ε is a constant of proportionality,alled absorbivity, b is path length, and c is the concentration

f the absorbing species, in this case concentration of the ascor-ic acid (mg/ml)). Applying this standardised relationship, theupernatant obtained during synthesis was analysed using UVpectrophotometry to determine the ascorbic acid concentration.

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he amount of the ascorbic acid not encapsulated was deter-ined from the supernatant’s concentration of the ascorbic acid

nd volume. Knowing the initial amount of the ascorbic acidsed in DLPLG/ascorbic acid nanoparticle synthesis, the per-entage of the ascorbic acid loaded into the nanoparticles wasbtained (loading efficiency). Loading amount was calculated42] by means of equation:

oading amount =[

Loading efficiency(%)

100

]

×Total amount of ascorbic acid added

.2.4. Degradation studiesTime of complete resorption of poly(dl-lactide-co-glycolide)

lactide/glycolide, 50/50) in the body is 4–8 weeks. The DLPLGarticles without and with encapsulated different concentrationf the ascorbic acid (DLPLG/ascorbic acid ratio was 85/15%,0/30% and 50/50%) have been sustained during 2 months8 weeks) in physiological solution at 37 ± 1 ◦C (Vaciotem P-electa). Na+ and Cl− ions are ions which dominate in thextracellular liquid and the composition of the physiologicalolution is 154 mM Na + 154 mM Cl− and its osmolarity cor-esponds with the osmolarity of the extracellular liquid. In theeriod of 2 months, the samples of the solution have been takenpproximately every week and analysed with UV spectroscopy.uring the experiment, the changes of the pH have been tracked

nd the samples have being taken regularly for examining thenfluence of the degradation on the morphological characteristicsf the particles.

.2.5. Infrared (IR) spectroscopyThe quality analysis of the samples has been performed

ith IR spectroscopy. The IR measurements were performed onerkin-Elmer 983 G Infrared Spectrophotometer, using the KBrellet technique, in the frequency interval of 400–4000/cm.

.2.6. Scanning electron microscope (SEM)The morphology of obtained particles of DLPLG without

nd with ascorbic acid has been examined by scanning electronicroscope JEOL JSM-649OLV. The powder samples for SEM

nalysis were coated with gold using the physical vapour depo-ition (PVD) process. Samples were covered with gold (SCD05 sputter coater), using 30 mA current from the distance of0 mm during 180 s.

.2.7. Stereological analysisThe particle size and morphology were examined using the

rea analysis method [43,44] by semi-automatic image anal-

w

ab

able 1oading efficiency and loading amount of DLPLG/ascorbic acid particles

Supernatant absorbance(264 nm)

Amount ofin supernata

LPLG/ascorbic acid 85/15% 0.0394 0.1581LPLG/ascorbic acid 70/30% 0.2493 0.6214LPLG/ascorbic acid 50/50% 0.7758 3.1000

s B: Biointerfaces 59 (2007) 215–223 217

ser (Videoplan, Kontron), connected with a scanning electronicroscope. From 200–300 particles in the SEM were measured

nd the following parameters were determined: area section Aa,erimeter Lp, maximal diameter of the particle Dmax, ferets xnd y, and form factor (fL).

.2.8. Ultraviolet (UV) spectroscopyThe UV measurements were performed on Perkin-Elmer

ambda 35 UV-vis Spectrophotometer in the frequency intervalf 200–400 nm.

.2.9. pH measurementsThe pH of the physiological solution has been measured using

H indicator strips obtained from Merck (KGaA, Germany)t various time periods to follow the acidity of the degradingedium with time.

. Results and discussion

The results of the determination of particle yield for variousLPLG/ascorbic acid ratios were similar and they were greater

han 50%. In the case of DLPLG/ascorbic acid 85/15% it was2.1%, in the case of DLPLG/ascorbic acid 70/30% it was 56.4%nd in the case of DLPLG/ascorbic acid 50/50% it was 52.8%.

Following the procedures from the above, the supernatantbtained from DLPLG/ascorbic acid nanoparticle synthesis wasnalysed to determine the amount of the ascorbic acid encapsu-ated within the nanoparticles. The results are shown in Table 1.he amount of the ascorbic acid in the supernatant has beenalculated from the product of the supernatant’s absorbancet 264 nm and its measured volume (25.5 ml). Assuming thatll of the ascorbic acid concentration not found in the super-atant was encapsulated by DLPLG nanospheres, the loadingfficiency was determined to be greater than 90% in all ratios ofLPLG/ascorbic acid nanoparticles.In order to investigate the structural characteristics of the

LPLG particles without and with encapsulated ascorbic acid.e. to confirm the quality composition of the samples IR spec-roscopy has been used. The IR spectra in Fig. 1 illustratell characteristic groups for copolymer poly(dl-lactide-co-lycolide). The IR spectra of DLPLG show peaks at 2961, 2943,841 (CH bend), 1760 (C O ester), 1440, 1420, 1310 (CH3),139, 1060, 978 (C O stretch), 768, 506 (CH bend)/cm. The IRpectra, also, shows a broad band in the range 3100–3600/cm

hich belongs to the OH− group of the water molecule [45,46].Comparing the obtaining IR spectra for DLPLG and ascorbic

cid (Fig. 2) with the characteristics IR spectra for ascor-ic acid shown in the literature [47,48] it is confirmed that

ascorbic acidnt (mg)

Loading efficiency(%)

Loading amount(mg)

98.2 8.641097.1 20.798693.8 46.9000

218 M. Stevanovic et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 215–223

FD

Fig. 1. IR spectra of the DLPLG nanospheres.

ig. 3. SEM images of (a) DLPLG nanospheres and nanoparticles with differentLPLG/ascorbic acid 70/30% and (d) DLPLG/ascorbic acid 50/50%.

Fig. 2. IR spectra of the DLPLG/ascorbic acid 85/15 nanoparticles.

ratio of DLPLG and ascorbic acid, (b) DLPLG/ascorbic acid 85/15%, (c)

M. Stevanovic et al. / Colloids and Surface

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0.32

0.98

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s B: Biointerfaces 59 (2007) 215–223 219

btained nanoparticles are composed of poly(dl-lactide-co-lycolide) and ascorbic acid. Besides the characteristic groupsor copolymer DLPLG the four O-H bands of ascorbic acidould be assigned by means of infrared investigations at 3529,420, 3316, 3215 cm−1. The spectra show bands that can bessigned to CH3, CH2 or CH groups in the ascorbic acid envi-onment at 2720 cm−1 and the spectra also clearly show theand corresponding to C O groups at 2916 cm−1. The bandshat correspond to the wave number 1750 cm−1, 1672 cm−1 and

021 cm−1 belong to C C, C-O-C and C-O groups, respectively.

The morphological characteristics of the DLPLGanospheres without and with encapsulated ascorbic acid

ig. 4. Comparative results of the stereological examining of (a) DLPLGanospheres and nanoparticles with different ratio of DLPLG and ascorbiccid, (b) DLPLG/ascorbic acid 85/15%, (c) DLPLG/ascorbic acid 70/30%, (d)LPLG/ascorbic acid 50/50%, based on feret x.

ig. 5. Comparative results of the stereological examining of (a) DLPLGanospheres and nanoparticles with different ratio of DLPLG and ascorbiccid, (b) DLPLG/ascorbic acid 85/15%, (c) DLPLG/ascorbic acid 70/30%, (d)LPLG/ascorbic acid 50/50%, based on perimeter form factor, fL.

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20 M. Stevanovic et al. / Colloids and Sur

ave been examined by SEM. From the Fig. 3a, it can beoticed the DLPLG particles have spherical shapes, theyre very uniform and non-agglomerated. Their mean sizesre 110 nm according to the stereological parameter feret y.or DLPLG/ascorbic acid 85/15% nanoparticle, as shown inig. 3b, the particles are also spherical, non-agglomeratednd exceptionally uniform, but their dimensions are slightlyigger than dimensions of blank DLPLG particles. In casef DLPLG/ascorbic acid 70/30%, Fig. 3c, it can be seen theimensions are increased, but the particles are still spherical. Inase of DLPLG/ascorbic acid 50/50%, Fig. 3d, the uniformitiesre compromised, the dimensions are increased, and thearticles have both spherical and irregular shapes with a lot ofgglomeration present.

The parameter which characterises the size (area section, Aa;

erimeter, Lp; maximal diameter of the particle, Dmax; feret xthe projection of the particle on x axis) and feret y (the pro-ection of the particle on y axis)) and the shape (form factor,L) of the particles are set based on the stereological analysis.

sf70

ig. 6. UV spectra for (a) DLPLG, (b) DLPLG/ascorbic acid 85/15%, (c) DLPLG/asf the degradation.

B: Biointerfaces 59 (2007) 215–223

or all parameters, minimum, maximum and mean values wereecorded and presented in Table 2.

Based on the obtained results of the stereological analysis ofLPLG particles, it is visible that they are uniform, their aver-

ge mean size varies from 0.11 to 0.17 �m depending of thetereological parameter taken in consideration (feret x, feret yr Dmax) (Table 2). Feret x values range from 0.05 to 0.22 �mith its mean size 0.12 �m (Fig. 4). Fig. 5 presents comparative

esults of DLPLG particles with and without ascorbic acid basedn their perimeter form factor. From the comparative resultsf the stereological analysis of the perimeter form factor (fL)Fig. 5) we can see DLPLG particles without ascorbic acid havehe highest mean value of perimeter form factor which is 0.90.anoparticle DLPLG/ascorbic acid 85/15% has minimum feretof 0.09 �m and maximum feret x of 0.46 �m where its mean

ize is 0.20 �m (Fig. 4). The mean value of the perimeter formactor is 0.87 (Fig. 5). For nanoparticle DLPLG/ascorbic acid0/30%, minimum feret x is 0.10 �m and maximum feret x is.87 �m where its mean size is 0.38 �m (Fig. 4) which indicates

corbic acid 70/30%, (d) DLPLG/ascorbic acid 50/50%, after different periods

M. Stevanovic et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 215–223 221

Fig. 7. Comparative curves for the dependence of the absorbance maximumfrom the time of the degradation for the DLPLG without and with ascorbic acid.

Fig. 8. Comparative cumulative curves of the release of the ascorbic acid inpercentages over the period of time of the degradation.

Fig. 9. SEM images of (a) DLPLG nanospheres and (b) DLPLG/ascorbic acid 85/15%, (c) DLPLG/ascorbic acid 70/30% and (d) DLPLG/ascorbic acid 50/50%particles, after 2, 24 and 39 days of the degradation.

2 faces B: Biointerfaces 59 (2007) 215–223

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22 M. Stevanovic et al. / Colloids and Sur

hat the uniformity is decreased and size increased. The meanalue of the perimeter form factor is 0.83 (Fig. 5). For nanoparti-le DLPLG/ascorbic acid 50/50%, minimum feret x is 0.25 �mnd maximum feret x is 4.17 �m where its mean size is 1.24 �mFig. 4). The mean value of the perimeter form factor is 0.76Fig. 5).

The pace of degradation of the DLPLG nanospheres with-ut and with encapsulated ascorbic acid as well as tracking theelease of the ascorbic acid from the polymeric matrix during theegradation process, have been examined with UV spectroscopy.he degradation of the DLPLG and release of the ascorbic acidave been tracked based on the intensity of the absorbance max-mum which is in the correlation with the concentration of theLPLG and ascorbic acid within the solution. The characteris-

ic absorbance peak which belongs to the origin, blank DLPLGample is at 270 nm. This absorbance peak increases with time ashe glycolide content increases in the degradation medium. Fig. 6hows the UV spectra obtained from measuring absorbance inhe supernatant for the blank DLPLG and DLPLG with variousoncentrations of the encapsulated ascorbic acid after differenteriods of the degradation, i.e. after two, 11, 17, 24, 31, 39, 46nd 55 days. Fig. 7 shows comparative curves for the dependencef the absorbance maximum from the time of the degradationor the DLPLG without and with ascorbic acid.

The UV spectroscopy has been used to estimate the concen-ration of the released ascorbic acid encapsulated in the polymerarticles. A calibration curve of ascorbic acid in physiologi-al solution at different concentrations has been prepared usinghe specific absorbance peak of ascorbic acid at 264 nm. Thisbsorbance is correlated with the calibration curve and amountf ascorbic acid is determined in percentages. Fig. 8 gives com-arative cumulative curves of the release of the ascorbic acidn percentages over the period of time of the degradation. Inhe first 24 days of the degradation, for all samples, less thanhe 10% of the encapsulated ascorbic acid have been released.or all DLPLG/ascorbic acid samples, the overall quantities of

he encapsulated ascorbic acid have been released in 8 weeks ofhe degradation. By the end of the experiment, the nanoparticlesave fully degraded and there were no more traces of them inhe solution.

The different ionised forms of the ascorbic acid have differ-nt redox properties, so that the redox-chemistry of the ascorbiccid is highly pH dependent [49–51]. Ascorbic acid decomposesnto biologically inactive compounds by auto-oxidation only atlkaline pH [52]. In the solution with low pH, decompositionf the ascorbic acid can happen for example under the influencef the enzymes (enzymatic oxidation) [52]. The UV spectra ashown in Fig. 6(b)–(d) is characteristic for the ascorbic acidhile the absorption maximums which would correspond to,3-diketo-l-gulonic acid (the first product of the ascorbic acidecomposition) have not been noticed, so there is no evidenceor the conversion of the ascorbic acid into 2,3-diketo-l-guloniccid.

The effect of degradation on the morphology of the DLPLGarticles without and with ascorbic acid has also been exam-ned. Scanning electron microscope has been employed to asseshe changes in the sample morphology with degradation. Scan-

4

a

ig. 10. Changes in the pH of the physiological solution with immersion time forhe DLPLG particles without and with different concentration of ascorbic acid.

ing electron micrographs of the dry degraded DLPLG particlesithout and with ascorbic acid are shown in Fig. 9. In the start-

ng phases of the degradation, a small reduction of the particleizes was noticeable. As the polymeric matrix degrade, the sizesf the particles decreases followed by intensive release of thescorbic acid. The particles are in a very close contact duringhe degradation process which brings to higher agglomerationf the particles and creation of the porous film. The samplesere examined after 2, 24 and 39 days of the degradation. Afterdays of the degradation, all samples have preserved the initial

hape of the particles, after 24 days signs of the agglomerationere visible, and after 39 days porous film has been created.

t is assumed that degree of porosity will be increased with theegradation time until the complete degradation of the samplesithin the solution.Fig. 10 shows the changes in the pH of the physiological solu-

ion with immersion time for the blank DLPLG particles and forLPLG particles with different content of ascorbic acid. The pHf the solution began to decrease after 2 days of immersion. After5 days of immersion, the pH of the physiological solution hadropped from pH 7.0 to 3.6 in the case of blank DLPLG, from pH.0 to 2.6 in the case of the DLPLG/ascorbic acid 85/15%, fromH 7.0 to 2.3 in the case of the DLPLG/ascorbic acid 70/30%,nd from pH 7.0 to 1.8 in the case of the DLPLG/ascorbic acid0/50%. DLPLG degrades via backbone hydrolysis (bulk ero-ion) and the degradation products are the monomers, lactic acidnd glycolic acid. It could be expected that the faster degradationf the lower molar mass fraction, present in copolymer, increasehe local acidity, thereby, accelerating the hydrolysis of higher

olar mass species. In other words, when acid accumulationreates a local pH drop, catalytic degradation of the polymertself occurs.

. Conclusion

The chemical method solvent/non-solvent has produced non-gglomerated DLPLG nanospheres with dimensions in the range

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M. Stevanovic et al. / Colloids and Su

rom 110 to 170 nm depending of the stereological parameteraken in consideration (feret x, feret y or Dmax), with sphericalnd uniform shapes. The ascorbic acid has been successfullyncapsulated into nanospheres thus creating nanoparticles witharious morphological characteristics depending of the concen-ration of the ascorbic acid. The degradation of the DLPLGithout and with ascorbic acid within the physiological solu-

ion have been tracked for 8 weeks and it has been determinedhat DLPLG completely degrades within this period fully releas-ng all the encapsulated ascorbic acid. In the first 24 days, theamples degrade slower while later the pace of the degrada-ion increases. In the first 24 days of the degradation, for allamples, less than 10% of the encapsulated ascorbic acid haveeen released. At the beginning the particles maintain the initialhape, but after 24 days the particles start being agglomerated,reating the porous film, where the porosity increases until theomplete degradation of the samples.

cknowledgements

Authors would like to thank Zoran Nedic and Milos Bokorovor their assistance in IR and SEM analysis. The Ministry ofcience and Environmental Protection of Republic of Serbiaupports this work through the project no. 142006.

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