FABAD J. Pharm. Sci., 29, 111-116, 2004 RESEARCH ARTICLE
IInnfflluueennccee ooff A Acccceelleerraatteed d SSttoorraaggee C Coonnd diittiioonnss oonn tthhee SSttaabbiilliittyy ooff V Vaannccoom myycciinn--L Looaad deed d PPoollyy((D D,,L L--llaaccttiid dee--ccoo-- ggllyyccoolliid dee)) M Miiccrroosspphheerreess
Burcu SAYIN*, Sema ÇALIfi*°
Influence of Accelerated Storage Conditions on the Stability of Vancomycin-Loaded
Poly(D,L-lactide-co-glycolide) Microspheres
Summary
Poly(lactide-co-glycolide) (PLGA) polymers are widely used synthetic biodegradable materials, especially for controlled deli- very of drugs, but conditions affecting degradation mechanisms of these polymers have not been studied in detail in microsphere systems. In this study, the effect of temperature and humidity on the stability and physicochemical characteristics of vancomycin- loaded PLGA (75:25) (MW 136,000) microspheres was investi- gated. The changes in the surface morphology, drug content and particle size distribution of the microspheres prepared by the o/w type emulsion solvent evaporation method were examined and evaluated after a three-month storage under accelerated conditi- ons (40 ± 2°C, 75 ± 5% relative humidity). At the end of this period, it was observed that the spherical shape of the microsp- heres was completely deformed and that polymer particles had formed on the surface, the particle size of the microspheres had increased from 77 µm to 810 µm, and almost 10% of vancomy- cin content was determined to be lost.
K
Keeyy WWoorrddss :: PLGA, stability, microspheres, temperature, hu- midity, accelerated conditions.
Received : 1.4.2005 Revised : 2.5.2005 Accepted : 3.5.2005
Vankomisin Yüklü Poli(D, L-laktid-ko-glikolid) Mikrokürelerinin Stabilitesi Üzerine H›zland›r›lm›fl
Saklama Koflullar›n›n Etkisi
Özet
Poli(laktid-ko-glikolid) (PLGA) polimerler; özellikle ilaçlar›n kontrollü tafl›nmas›nda s›kl›kla kullan›lan sentetik biyoparçala- nabilir materyallerdir, ancak literatürde mikroküre sistemleri için bu polimerlerin degradasyon mekanizmalar›n› etkileyen koflullarla ilgili incelemeler çok fazla bulunmamaktad›r. Bu ça- l›flmada, vankomisin yüklü PLGA (75:25) (MA 136,000) mik- rokürelerinin stabilite ve fizikokimyasal özellikleri üzerine s›cak- l›k ve nemin etkisi incelenmifltir. Y/S emülsiyon-çözücü buhar- laflt›rma yöntemi kullan›larak haz›rlanan mikroküreler h›zlan- d›r›lm›fl koflullar alt›nda (40 ± 2°C, %75 ± 5 relatif nem) 3 ay boyunca saklanarak, yüzey özellikleri, ilaç içeri¤i ve partikül büyüklü¤ü da¤›l›m›ndaki de¤ifliklikler izlenmifltir. Bu sürenin sonunda, mikrokürelerin küresel yap›s›n›n tamamiyle bozuldu-
¤u ve polimer parçalar›n›n olufltu¤u, partikül büyüklü¤ünün 77 µm’den 810 µm’ye yükseldi¤i ve vankomisin miktar›nda yakla- fl›k %10 azalma oldu¤u görülmüfltür.
A
Annaahhttaarr KKeelliimmeelleerr :: PLGA, stabilite, mikroküre, s›cakl›k, nem, h›zland›r›lm›fl koflullar.
IINNTTRROODDUUCCTTIIOONN
Poly(lactide), poly(glycolide) and their copolymers approved by the U.S. Food and Drug Administrati-
on (FDA) represent a major class of synthetic bi- odegradable materials with wide medical applicati- ons in areas such as wound closure, controlled rele- ase systems, orthopedics and tissue engineering.
* Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara-TURKEY
° Corresponding author e-mail: scalis@hacettepe.edu.tr
namely surface erosion and bulk erosion6. The poly- mer chains undergo bulk degradation and the deg- radation occurs at a uniform rate throughout the PLGA matrix7. The degradation rate of the PLGA copolymers is dependent on the molar ratio of the lactic and glycolic acids in the polymer chain, mole- cular weight of the polymer, the degree of crystalli- nity and the Tg of the polymer7. Glycolide content in the copolymer structure increases the degradation rates and the increase in degree of crystallinity is fo- und to retard hydrolytic degradation, but only to a certain extent8. Previously, it was observed that the high temperatures applied to polylactides caused shortening of the degradation time9. The PLGA polymer biodegrades into toxicologically acceptable lactic and glycolic acids that are eventually elimina- ted from the body through the Krebs cycle4,10.
During the stability studies on the microspheres, surface morphology, particle size and differences in drug content values were investigated.
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MAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS
M Maatteerriiaallss
Vancomycin was donated from Eli Lilly (Turkey).
PLGA (75:25) was provided by Merck Dupont (Cin- cinnati, IL, USA). Sodium oleate (SO) was obtained from Aldrich (Germany). Polyvinyl alcohol (PVA) was purchased from Sigma (USA). Methylene chlo- ride and dimethyl sulfoxide were supplied by Merck (Germany). All other chemicals used were of analytical reagent grade.
M Meetthhooddss
PPrreeppaarraattiioonn ooff vvaannccoommyycciinn--llooaaddeedd PPLLGGAA mmiiccrroosspp-- hheerreess
Vancomycin-loaded PLGA microspheres were pre- pared by a modified o/w type emulsion/solvent evaporation process as described before11,12. Briefly, vancomycin was dissolved in dimethyl sulfoxide and added to the polymer solution (PLGA 75:25) in methylene chloride. Then, this dispersion was emul- Say›n, Çal›fl
The reason for the wide range of use of these synthe- tic polymers is their resorbability through natural pathways, low toxicity, satisfactory mechanical strengths, biodegradability and biocompatibility. In addition, these copolymers have attracted much at- tention because their biodegradation rate can easily be controlled by altering its composition. They offer a broad range of systems with a rate of delivery that can be modulated over the required time period, for example from days to several weeks to over a year.
Microparticulate controlled-release systems, prepa- red from aliphatic polyesters such as poly(D,L-lacti- de-co-glycolide) (PLGA), have been widely investi- gated over the past decades for the delivery of drugs1-4. Many studies have been conducted on the- ir biological, physical and chemical properties, alt- hough little is known about the stability of the poly- mer formulations in solid state during storage at de- termined temperatures or humidity conditions. It is a well-known fact that for drug delivery systems, stability of the formulation is one of the most critical parameters from the pharmaceutical aspect. The sto- rage conditions are particularly important to define in order to start biological studies and to make sure that the drug doses used would be preserved. For this purpose, accelerated stability testing at high temperatures and humidity conditions are often employed to predict the shelf life of drugs.
PLGA and PLLA (poly-l-lactic acid) are both hydrolytically unstable, and although insoluble in water, they degrade by hydrolytic attack of their es- ter bonds. The main mode of degradation for the PLGA polymer is purely through simple hydrolysis of the ester bonds and does not involve any enzyma- tic activity. In vivo it degrades into lactic acid and glycolic acid. Lactic acid enters the tricarboxylic acid cycle and is metabolized and subsequently elimina- ted from the body as carbon dioxide and water.
Glycolic acid is either excreted unchanged in the kidney or it enters the tricarboxylic acid cycle and is eventually eliminated as carbon dioxide and water5. Polymer erosion in microparticles is the degradation of the polymer to water-soluble fragments, accom- panied by progressive weight loss of microparticles.
In general, two erosion mechanisms are described,
FABAD J. Pharm. Sci., 29, 111-116, 2004
sified into the aqueous continuous phase containing polyvinyl alcohol:sodium oleate (PVA:SO). This me- dium was stirred continuously (750 rpm) at room temperature for two hours. Finally, microspheres were collected by centrifugation, washed in water and dried at room temperature.
SSttaabbiilliittyy ssttuuddiieess oonn mmiiccrroosspphheerreess
The stability of vancomycin-loaded microspheres was followed under accelerated conditions (40±2°C and 75±5% relative humidity) for a three-month pe- riod. At regular intervals (3rd day, and 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, and 12th weeks), charac- terization parameters such as surface morphology, drug content and particle size distribution of the microspheres were examined and evaluated.
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Miiccrroossccooppiicc eevvaalluuaattiioonn
Scanning electron microscopy (SEM) was used to examine the surface characteristics of the microsp- heres during the stability studies. For morphological examination under SEM, microspheres were moun- ted on metal stubs with conductive silver paint and then sputtered with a 150°A layer of gold in a Bio- Rad (England) sputter apparatus. Samples were in- vestigated in Jeol SEM at 80 kV (SEM ASID-10 Devi- ce, Japan).
P
Paarrttiiccllee ssiizzee aannaallyyssiiss
The particle size distribution of the microspheres was measured using a HELOS laser diffraction type particle size analyzer* (Sympatec, Germany). Before the measurement process, microspheres were dis- persed in purified water containing 0.1% Tween 80 as a dispersing agent. Each measurement was the mean of three samples.
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Miiccrroosspphheerree ddrruugg llooaaddiinngg
In order to determine the drug loading value, van-
comycin-incorporated microspheres were accura- tely weighed and dissolved in methylene chloride.
Afterwards, pH 7.4 phosphate buffer was added and extracted for three hours. After the evaporation of methylene chloride, the polymer was removed, the solution was filtered (Swinnex-GS, Millipore, UK) and vancomycin content was detected by vali- dated UV spectrophotometric method (Shimadzu, Japan) at 280 nm.
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REESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN
From a general point of view, the main phenomena discussed about the degradation process of PLGA polymer is that degradation causes an increase in the number of carboxylic end groups, which are known to autocatalyze ester hydrolysis13. Polylacti- de (PLA) and poly-(lactide-co-glycolide) (PLGA) are moisture- and heat-sensitive polymers6. In this study, vancomycin-incorporated microparticles sto- red under accelerated conditions in a climate cabinet showed significant aggregation and degradation af- ter 12 weeks, although SEM of particles taken after eight weeks’ storage showed that no loss in shape or coalescence of the particles had occurred during the induction period under these conditions. At the end of the 10th week, visible deformation was observed on the surface morphology and spherical shape of the microspheres appeared to change, whereas at the 12th week, the microspheres seemed to deform completely and the polymer particles had formed on the surface (Fig. 1). The visible deformation determi- ned on the surface morphology and the variation in spherical shape were considered to be caused by hydrolytic degradation of PLGA polymer after three months of storage under accelerated conditi- ons. It has been shown previously that the degrada- tion rate is affected by several physical and chemical factors, such as initial pH, ionic strength and tempe- rature of external medium, copolymer ratio, molecu- lar weight, crystallinity, and specimen size14,15. The temperature dependence of the PLGA degradation rate has not been systematically studied, but faster
*Particle size analysis was performed in Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Technology.
Say›n, Çal›fl
ge molecular weight15. On the contrary, in another report, visual examination of the stored microsphe- res prepared with cellulose propionate polymer re- vealed no significant changes in the physical appe- arance of the microspheres after storing them at 40°C/75% relative humidity for 10 weeks19.
It was found that a linear relationship between the degradation rate and particle size existed, with the larger particles degrading fastest16. In this study, the particle size of the prepared microspheres was app- roximately 77 µm microspheres. The particle size of the microspheres was determined to increase signi- ficantly from 77 µm to 810 µm after 12 weeks (Table 1, Fig. 2). This situation suggested that the humidity accelerated the aggregation of the microspheres and
for this reason particle sizes of the microspheres we- re determined to increase. Furthermore, polymer degradation might have caused an increase in par- ticle size, which was determined to a significant ex- tent in SEM photographs after 10 weeks’ storage. It has also been reported recently that PLGA (50:50) nanoparticles of an average particle size below 80 nm exhibited a significant increase in particle size, suggesting an aggregation process after six months of storage at room temperature. On the other hand, neither the mean particle size nor the size distributi- on profile of PLGA (50:50) nanoparticles was affec- ted when stored at 8°C17.
degradation of poly(lactic acid) and poly(glycolic acid) homo- and co-polymers at elevated temperatu- re has been reported16. During this study, it was ob- served that the drug degradation increased with the elevation of temperature. It was reported in literatu- re previously that the lower the initial molecular we- ight of the polymer, the lower the molecular weight variation after storage17. The polymer molecular we- ight not only translates to longer degradation times but also to a change in polymer properties that may have an effect on water diffusivity or polymer deg- radation18. When preparing our microspheres, a high molecular weight PLGA polymer (75:25, MW 136,000) was selected for implant formulations. It has been reported that the initial mass loss and the amount of water absorbed were functions of avera- FFiigguurree 11.. SEM photographs of the vancomycin-loaded PLGA microspheres following the stability stu-
dies under accelerated conditions. FFiigguurree 22.. Particle size change of microspheres following the stability study realized under accelerated conditions (n = 3).
INITIAL
Particle size (µm)
INITIAL
AFTER 2 WEEKS AFTER 4 WEEKS
AFTER 6 WEEKS AFTER 8 WEEKS
AFTER 10 WEEKS AFTER 12 WEEKS
FABAD J. Pharm. Sci., 29, 111-116, 2004 T
Taabbllee 11.. Particle size change and vancomycin amount determined in microspheres fol- lowing the stability study realized under accelerated conditions
SSaammppllee TTiimmee PPaarrttiiccllee ssiizzee VVaannccoommyycciinn aammoouunntt ((µµmm)) nn == 33 ddeetteerrmmiinneedd iinn
m
miiccrroosspphheerreess ((%%))
Initial 77.33 100.00
3rd day 103.66 99.60
1st week 109.30 98.90
2nd week 114.68 98.30
3rd week 112.73 97.80
4th week 167.99 98.50
5th week 120.07 93.70
6th week 166.67 96.88
7th week 233.36 96.58
8th week 231.88 95.41
10th week 474.25 91.47
12th week 810.91 91.11
In another previous study, the effect of change in dissolution rate as a function of storage on PLGA microspheres prepared by an o/w type emulsi- on/solvent evaporation technique using mechanical agitation was investigated, and accelerated degrada- tion of PLGA was exhibited and a substantial chan- ge in release of drug was shown after three months of storage at 40°C. In addition, multiphase microsp- heres of the W/O/O/O type were found to be stab- le20. In another report, under prolonged storage up to 90 days, especially at 45°C, temperature became a dominant factor causing an increase in drug release from diclofenac wax microspheres21.
According to a previous paper reporting on DL-PLA microspheres of different molecular weights prepa- red by the solvent evaporation method, size, shape, and loading did not change after a three-year stora- ge at room temperature (20 ± 2°C) under desiccated conditions22. DL-PLA polymer is more hydrophobic than the glycolide polymer because of the methyl group present in its structure and has less crystalli- nity. Crystallinity and water uptake are key factors in determining the rates of degradation10. Therefore, formulations prepared with PLGA polymer seem to
have the fastest degradation rate. Only after a criti- cal degree of degradation is reached do the poly- mers form a network of pores that allows for the re- lease of monomers and oligomers18.
At the end of the 3rd month of the stability study, al- most 10% of vancomycin was determined to be lost (Table 1, Fig. 3). Manufacturers recommend the sto- rage conditions of vancomycin as 2-8°C23. In additi- on, it was also reported in the literature that the pre-
pared admixture with 5% dextrose and 0.9% sodium chloride can be stored in a refrigerator for 96 hours without significant loss of potency24. At 66°C, van- comycin dissolved in phosphate buffer and 5% dext- rose solution, in which the antibiotic degraded in an apparent first order fashion25. It was recommended that all of the preparations containing vancomycin must be stored at room temperature in tightly sealed containers26.
CCOONNCCLLUUSSIIOONNSS
As a result of this study, it was concluded that the storage temperature and humidity appeared to af- fect the stability of the vancomycin-loaded PLGA microspheres. A temperature of 40±2°C and relative humidity of 75±5% caused instability signs in the samples. After a three-month storage under accele- rated conditions, aggregation and degradation were detected in microspheres, the particle size of the FFiigguurree 33.. Vancomycin amount determined in microsphe- res following the stability study realized under accelerated conditions (n = 3).
% Remaining Vancomycin Amount
Say›n, Çal›fl
microspheres was increased, and drug amount was determined to decrease with 10% drug loss. In conc- lusion, it was clear that the stability of the polymeric system widely depended on the temperature and humidity conditions of the storage.
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Acckknnoowwlleeddggeemmeenntt
This study was supported by Hacettepe University Research Fund (Project No: 0201301003). The aut- hors are very grateful to Associate Professor Musta- fa Sargon (MD) for carrying out the electron micros- copic analysis. The authors also wish to acknowled- ge Dupont Merck (USA) for the generous supply of PLGA and Eli Lilly (Turkey) for vancomycin.
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