Current Drug Delivery, 2006, 3, 343-349 343

The Effect of Different Grades of PLGA on Characteristics of Micro-

spheres Encapsulated with Cyclosporine A

Bizhan Malaekeh-Nikouei 1, Sayyed A. Sajadi Tabassi2,*, Mahmoud R. Jaafari3

1
Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Research Centre, Mashhad University of
Medical Sciences, Mashhad, Iran; 2Department of Pharmaceutics, School of Pharmacy and Pharmacological Research
Center for Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran; 3Department of Pharmaceutics,
School of Pharmacy and Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract: The aim of this study was to evaluate the effect of different grades of poly D, L lactide-co-glycolide (PLGA) on
the properties of microspheres encapsulated with Cyclosporine A (CyA). Microspheres were prepared by solvent evapo-
ration method using three grades of PLGA. Various characteristics of microspheres such as morphology, size distribution,
encapsulation efficiency and release profile were evaluated. Complementary studies were also carried out by Infrared (IR)
spectroscopy and Differential scanning calorimetry (DSC) to evaluate possible drug-polymer interactions. Scanning elec-
tron microscopy (SEM) studies showed microspheres as spherical particles with CyA deposited as islands on the surface
of spheres. Particle size range was 1-25 µm for microspheres made of PLGA (50:50) which showed the minimum size.
Encapsulation efficiency was found to vary from 75% to 92% in various formulations. The profile of release was biphasic,
showing an initial rapid phase followed by a continuous and slower rate thereafter. Microspheres made of grades 50:50
and 85:15 showed the highest and lowest amount of drug release, respectively. IR spectra for drug, polymer and micro-
spheres did not indicate any chemical interaction between the components of microsphere and DSC thermograms revealed
that CyA was present in its amorphous state within microspheres. In conclusion, the effect of polymer characteristics
should be considered in microsphere formulations. In this study, suitable microspheres especially with PLGA (50:50)
were prepared which allow the controlled release of CyA over a prolonged period of time.
Keywords: Biodegradable, cyclosporine A, microsphere, PLGA, release.

INTRODUCTION The use of biodegradable microspheres as drug delivery

systems offers several important advantages including con-
Cyclosporine A (CyA) is a highly lipophilic oligopeptide
trolled drug release, ease of administration through regular
with immunosuppressant properties which is used to prevent intramuscular or subcutaneous injections, biodegradability
organ rejection after transplantation [1, 2]. This drug is a and biocompatibility [17, 18]. Advantages of microspheres
highly specific and potent inhibitor of T-lymphocytes which
for oral administration of drugs are well established as slow
is also administered in the treatment of selected autoimmune
and continuous release, protection of drug against acid and
diseases such as rheumatoid arthritis, Behcet’s disease, pso-
enzymes in the GI tract [9]. After subcutaneous or intramus-
riasis and nephrotic syndrome [1, 3, 4, 5, 6]. Administration
cular administration of microspheres, the loaded drugs are
of this drug is limited by its toxic effects to liver, kidney and
directly reaching the tissue of interest or the general circula-
nervous system [7]. Due to relatively high molecular weight,
tion or they act as a reservoir to release the drug over a pro-
very high lipophilicity and poor solubility in aqueous media,
longed period of time [19].
oral absorption of CyA is usually very low [7, 8]. With con-
ventional delivery systems, bioavailability after oral intake Recently, several studies have been carried out for prepa-
varies from 20 to 50% [9]. Blood levels around the thera- ration of microspheres containing CyA. In 1993, CyA loaded
peutic window may also cause adverse effects such as neph- PLGA microparticles were prepared with a biphasic in vitro
rotoxicity, neurotoxicity and hypertension and low levels of release profile [15]. In another study, effects of polymer
CyA could be associated with the risk of organ rejection amount and stirring rate on the size, morphology and encap-
[5, 8]. sulation efficiency of CyA loaded PLGA microspheres were
investigated [20]. Modification of CyA release from PLGA
To overcome the problems associated with conventional
microspheres by adding fatty acid esters was evaluated [6].
delivery systems, alternative dosage forms such as liposomes Also, CyA loaded microspheres and nanospheres were pre-
[7, 10, 11], microemulsions [12], microspheres [3, 13, 14,
pared using polycaprolactore [21], dextrin [7] and stearic
15] and nanospheres [13, 16] have been suggested to in-
acid [22].
crease the therapeutic efficacy of CyA and also to reduce its
adverse effects. The aim of this study was to evaluate the effect of differ-
ent grades of PLGA on characteristics of microspheres en-
capsulated with CyA to help for pharmaceutical design of
*Address correspondence to this author at the School of Pharmacy, Mash-
had University of Medical Sciences, P.O. Box 91775-1365, Mashhad, Iran;
microsphere formulations in the future. With regard to the
Tel: 98 511 8823255; Fax: 98 511 8823251; E-mail: s-sajjadi@[Link] lipophilicity of CyA and characteristics of PLGA such as

1567-2018/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

344 Current Drug Delivery, 2006, Vol. 3, No. 4 Malaekeh-Nikouei et al.

biocompatibility and biodegradability [20, 21], this polymer shaken in a water bath at 37 °C. At various time intervals,
was chosen. According to the effect of molecular weight and aliquots (400µl) were taken after centrifugation (13000 rpm
lactide to glycolide ratio on properties of PLGA micro- for 5 min) and replaced with fresh medium. CyA released
spheres, in this investigation, three grades of PLGA differed into the supernatant was quantified by HPLC as described
in average molecular weight; lipophilicity and lactide to gly- above and cumulative release pattern was established and the
colide ratio were used. Complementary studies were also results were reported as Means ± SEM of three experiments
conducted using IR and DSC to investigate physical proper- [24].
ties of CyA within microspheres.
Differential Scanning Calorimetry (DSC)
MATERIALS AND METHODS
DSC was carried out using a DSC apparatus equipped
Materials with stare software (METTLER TOLEDO SW7.01, Swit-
PLGA 50:50 (Mw 40000-75000), PLGA 65:35 (Mw zerland). Thermograms of different samples including CyA,
40000-75000) and PLGA 85:15 (Mw 50000-126000) were PLGA and CyA loaded microspheres were obtained on 5-10
obtained from Sigma (USA). CyA was purchased from LC mg of samples put in sealed aluminum crucibles and heated
laboratories (USA). Dichloromethane and polyvinyl alcohol from 25 to 250 ºC at a rate of 10 ºC/min in a nitrogen atmos-
(PVA) (Mw 27000) were supplied by Merck (Germany). All phere. Empty 40 µl crucibles were used as reference.
materials were of analytical grade unless otherwise stated.
Infrared Spectroscopy (IR) Studies
Preparation of PLGA Microspheres IR spectra of different samples including CyA, PLGA
CyA loaded microspheres were prepared by solvent and CyA loaded microspheres were recorded using a Uni-
evaporation method [22]. Briefly, CyA and PLGA (with 1:5 com sp 1100 (England) in 500 to 4000 cm-1 wavelength and
and 1:10 ratios) were dissolved in dichloromethane. This potassium bromide disks containing the samples were used.
solution was emulsified in 25 ml of an aqueous solution
containing 0.3% w/w poly vinyl alcohol (PVA) using a ho- Statistical Analysis
mogenizer (Ultraturax, IKA, Germany) at 21000 rpm for 5 One-way Analysis of variance (ANOVA) was used with
min and left to be stirred for 24 hours. The microspheres Instat software (GraphPad Software, Inc.) to assess the sig-
were collected by centrifugation at 15000 g for 15 min and nificance of the differences among various groups. In the
washed twice with water to remove any residue of PVA. case of significant F value multiple comparison Tukey test
Finally, microspheres were vacuum dried in a freeze drier was used to compare the means of different treatment
(Heto, DW3, Denmark). The whole procedure was repeated groups. Results with p<0.05 were considered to be statisti-
for three grades of PLGA in triplicates. cally significant.

Optical and Electron Microscopy RESULTS

The shape and surface characteristics of microspheres Microscopic Evaluation and Size Analysis of Micro-
were examined by optical microscope (OLYMPUS, Ger- spheres
many) and SEM (LEO, 1450 VP, England), respectively. Microspheres were spherical in shape. No drug crystals
Samples were prepared on stubs and coated by platinum. were observed in any of the samples. Different PLGA micro-
spheres had similar morphologic features. Scanning electron
Particle Size Analysis micrographs in (Fig. 1) reveal that islands of CyA were
Mean diameters and particle size distribution of micro- formed on the surface of microspheres. Also, the dimension
spheres were measured by particle size analyzer (Klotz, of these islands increased in the 1:5 ratio compared to 1:10
Germany) after suitable dilution with double distilled water. ratio due to increasing drug loading.
Results were reported as Means ± SEM.
Table 1 summarizes the mean size and encapsulation
Determination of Encapsulation Efficiency efficiency data of different formulations. Microsphere size
varied from 1 to 25 µm and size differences between groups
To determine encapsulation efficiency, a weighed
was significant (P<0.05). Most microspheres were smaller
amount of CyA loaded microspheres was dissolved in 1 ml
than 10µ in size and similar size distributions were seen in
dichloromethane and after filtration through 0.45 µm filter, both 1:5 and 1:10 drug to polymer ratios.
20 µl of this solution was injected into HPLC. The HPLC
system consisted of pump and UV detector. Separation was Encapsulation efficiency ranged from 78 to 92% in dif-
achieved by using a reversed phase column. The mobile ferent formulations. Encapsulation efficiency of PLGA
phase was methanol:water (90:10) with flow rate of 1.5 (50:50), PLGA(65:35) in 1:5 or 1:10 ratios were not signifi-
ml/min and the concentration of CyA was determined at 214 cantly different (P>0.05) but the difference was significant
nm using standard curve [23]. Results were reported as Mean for PLGA(85:15) (P<0.05) in both 1:5 and 1:10 ratios. How-
± SEM of three measurements. ever, the difference in 1:5 and 1:10 ratios was not significant
in any of the groups (P>0.05).
In Vitro CyA Release
Twenty mg PLGA microspheres loaded by CyA was In Vitro CyA Release
suspended in phosphate buffer (pH 7.4, 500µl) containing A) microspheres with 1:5 drug to polymer ratio: As it is
0.01% w/v of polysorbate 80. The suspension was gently indicated in (Fig. 2), the release profile is biphasic, with an
The Effect of Different Grades of PLGA Current Drug Delivery, 2006, Vol. 3, No. 4 345

I) II)

Fig. (1). Scanning electron micrographs of microspheres encapsulated with CyA. I) 1:5 drug to polymer ratio, II) 1:10 drug and polymer ratio.
Magnification of images is Ä3900 and Ä2400 in part (I) and in part (II), respectively.

Table 1. Mean Size and Encapsulation Efficiency of Different Microspheres Encapsulated with CyA

PLGA grade Drug to polymer ratio Mean size (µm) Encapsulation efficiency (%)

50:50 1:5 2.905 ± 0.005 78.42% ± 0.34

50:50 1:10 5.810 ± 0.034 76.48% ± 1.50

65:35 1:5 3.769 ± 0.006 87.71% ± 0.34

65:35 1:10 6.123 ± 0.022 87.48% ± 0.17

85:15 1:5 4.631 ± 0.030 86.73% ± 1.25

85:15 1:10 7.740 ± 0.022 92.33% ± 2.34

Fig. (2). Cumulative percent release of CyA from microspheres made from 1:5 drug to polymer. (Mean±SEM, n=3).

initial rapid phase during the first 5 days follows by a con- B) microspheres with 1:10 drug to polymer ratio: Re-
tinuous and slower rate thereafter. Amount of CyA released lease pattern of these microspheres was similar to the previ-
after 24 days were 22.11% ± 1.55 for PLGA (50:50), 23.48% ous microspheres (Fig. 3). Microspheres made from grades
± 1.40 for PLGA (65: 35) and 12.31% ± 0.94 for PLGA (85: 50:50 and 85:15 of PLGA showed the highest and lowest
15). amount of release, respectively.
346 Current Drug Delivery, 2006, Vol. 3, No. 4 Malaekeh-Nikouei et al.

Fig. (3). Cumulative percent release of CyA from microspheres made from 1:10 drug to polymer. (Mean±SEM, n=3).

DSC Studies oral delivery due to the possibility of being uptaken by the
lymphoid system M cells in peyer's patches [25, 26]. Based
Thermograms of PLGA, CyA and microspheres were
on the mechanism of drug effect (T-cell inhibition), direct
compared in (Fig. 4). PLGA and CyA had an endotherm at
uptake by the lymphoid system will improve the therapeutic
50 ºC and 130 ºC which in the thermogram of microspheres
effect of CyA. In the meantime, they can form a drug reser-
the endothermic peak in 50 ºC was only observed and that of
voir at the injection site that releases the bioactive substance
CyA was eliminated.
over several days up to several months after intramuscular
injection [18, 19].
IR Studies
The difference among the encapsulation efficiency of
IR spectrum of CyA displays an intense amide carbonyl PLGA (85:15) microspheres and other grades might be due
band at 1627 cm -1 while that of PLGA shows carbonyl band to its higher lipophilicity. Also, the fact that encapsulation
at 1750 cm -1 and alkyl band at 2930 cm -1 (Fig. 5). IR spectra efficiency was not affected by the amount of drug to polymer
of microspheres showed the typical bands of both drug and ratio shows that the polymer was adequate for loading CyA
polymer without any spectral shift or new band. in 1:5 ratio. In another study on microspheres made with two
different grades of PLGA, it was shown that encapsulation
DISCUSSION efficiency was independent of molecular weight [15].
Evaluation of different characteristics of microspheres As mentioned earlier, CyA release had a biphasic pattern.
indicates that suitable microspheres with reasonable encap- In the first four days microspheres released 7 to 17% and the
sulation efficiency were prepared. SEM studies confirmed release continued by a slower rate up to a month. In a study
the presence of part of CyA as islands on the surface of mi- conducted in 2004, drug release in CyA loaded PLGA
crospheres. According to the CyA release profile, it seems nanoparticles, prepared by high-pressure emulsification sol-
that the amount of drug deposited on the surface of micro- vent evaporation, was 30-45% in the first 8 hours [9]. Re-
spheres is not considerable and most of CyA is incorporated sults of our study and other previous studies which conferred
within the microspheres. On the basis of island dimensions, with the lipophilic nature of polymer and drug were appar-
encapsulation efficiency might be reduced by decreasing ently contradicted to the results of this recent study. In an-
drug to polymer ratio. other study carried out on CyA loaded nanoparticles only 3%
Comparison of the size of different microspheres showed and 4% of CyA was released after 20 and 120 min, respec-
that by increasing the molecular weight of PLGA, micro- tively [2]. Also, in a recent study on the effect of micro-
spheres would become larger. The co-polymer with high sphere size on drug release, biphasic release pattern was re-
molecular weight has probably led to an increase in the vis- ported with greater release in the first seven days followed
cosity of the internal phase during emulsification process and by slower and continuous release in the following days [17].
also greater particle size. Moreover it has been suggested The initial phase of release is due probably to CyA adsorbed
that polymer lipophilicity influences particle size [3] and on on the surface or diffused near the surface and the second
the basis of an earlier study, PLGA (85:15) with the highest release phase occurs by polymer erosion/degradation. Poly-
molecular weight and lipophilicity has made the largest mi- mer degradation rate is dependent on lactide to glycolide
crospheres [25]. ratio of polymer, crystalinity, molecular weight and hydro-
philicity [17].
Taking into account that 70% of microspheres were
smaller than 10 µm, these microspheres can be suitable for
The Effect of Different Grades of PLGA Current Drug Delivery, 2006, Vol. 3, No. 4 347

I)

II)

Fig. (4). DSC thermograms of A)PLGA(65:35) microsphere, B)CyA, C)PLGA(65:35). I) 1:5 drug to polymer ratio, II) 1:10 drug to polymer
ratio.

Elimination of endothermic peak of CyA in microsphere CONCLUSION

thermogram confirms the amorphous state of CyA in micro-
sphere. In other DSC studies, decreasing in Tg of PLGA was In this study, microspheres with high encapsulation effi-
seen and molecular dispersion of CyA in microspheres was ciency were prepared. They showed biphasic release pattern.
Microspheres made by PLGA (85:15) showed higher encap-
concluded. However, in our study no significant movement
in endotherm peak of polymer was seen. In another study sulation efficiency, larger particle size and slower release
which was done by MTDSC, Tg of PLGA in microspheres rate than the other two grades, whereas PLGA (65:35) and
without CyA and with 10 to 50% did not change and the PLGA (50:50) microspheres showed nearly similar proper-
ties. The adequate characteristics regarding microsphere size,
glass transition temperature corresponding to the peptide was
observed for loaded microspheres and finally, amorphous encapsulation efficiency and release profile could be attrib-
state of CyA in microspheres was concluded [14]. A com- uted to the PLGA (50:50) microspheres. On the basis of
plementary study by IR indicated no spectral shifts. Conse- variations in particle size, encapsulation efficiency, and re-
lease pattern, selecting PLGA grade is an important factor in
quently, the possibility of drug and polymer interaction can
be excluded. microsphere preparation and it should be considered in mi-
crosphere formulations.
348 Current Drug Delivery, 2006, Vol. 3, No. 4 Malaekeh-Nikouei et al.

A)

B)

C)

Fig. (5). Infrared spectra of A)PLGA(85:15), B)CyA, C)PLGA(85:15) microsphere.

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Received: October 19, 2005 Revised: December 12, 2005 Accepted: March 07, 2006