|Título/s:||In vitro release testing of PLGA microspheres with Franz diffusion cells|
|Autor/es:||Herrera, Laura C.; Defain Tesoriero, María Victoria; Hermida, Laura G.|
|Institución:||INTI-Química. Buenos Aires, AR|
|Palabras clave:||Péptidos; Drogas; Ensayos; Células; Difusión; Estabilidad; Acetatos; Soluciones; Polímeros; Biodegradabilidad|
| Ver+/- |
Dissolution Technologies | MAY 20126
In Vitro Release Testing of PLGA
Microspheres with Franz Diffusion Cells
Laura C. Herrera, María V. Defain Tesoriero, and Laura G. Hermida*
Centre of Research and Development in Chemistry, National Institute of Industrial Technology,
Avenida General Paz 5445 (B1650WAB), San Martín, Buenos Aires, Argentina
In the present study, two methods were used to evaluate the in vitro release of leuprolide acetate (LA) from
poly(lactide-co-glycolide) (PLGA) microspheres: Franz diffusion cells, typically referred to as “vertical diffusion cells” (VDC),
and rotating bottle apparatus (RBA), both modified with a dialysis membrane. This hydrosoluble peptide was chosen as a
model drug to study different possibilities of in vitro testing and analyze the variables that affect drug release, respecting
sink and physiological conditions. Microspheres were prepared with a conventional double emulsion–solvent
evaporation method using PLGA (50:50) with a relatively low molecular weight. Comprehensive stability tests for LA were
performed in the conditions used for in vitro release assays. In phosphate-buffered saline (PBS), LA showed no significant
degradation, but in an acidic medium, it degraded dramatically. The release profile of the delivery system was governed
mainly by diffusion as explained by the low molecular weight of the polymer and the high water solubility of the peptide.
The in vitro release profiles were triphasic in vertical diffusion cells and biphasic in the rotating bottle apparatus. The
release kinetics was enhanced in RBA with respect to VDC, probably because the constant movement of a suspension of
loose microspheres in a large volume and the large membrane area facilitated drug migration. The smoother, triphasic
profiles obtained with VDC can be explained by the partial confinement of microspheres, which is similar to the described
in vivo behavior of an injectable delivery system.
The advent of modified-release delivery systems brought the complex issue of in vitro release evaluation, which has not yet been fully solved. Many
devices and methods have been tested to clarify the
matter and set specifications. Because not all dosage
forms should fulfill the same requirements, this becomes
even more difficult. For microspheres, much has been
done, but there is still disagreement about the best in vitro
release testing method to apply. Drugs and polymers of
different natures, microsphere features, in vitro release
devices, receptor media, and sink conditions are some
of the issues that may be encountered during decision-
making. Many authors have analyzed and discussed the
suitability of different devices to perform the release tests
of prolonged-release systems (1, 2). For instance, USP
Apparatus 4, which is based on flow-through cells, was
successfully used to obtain release profiles of dexametha-
sone from long-term, modified-release formulations (3).
This is in accordance with USP recommendations about
the suitability of this equipment for delivery systems
containing drugs with limited solubility (4). Dialysis tests
performed with different devices may be useful for testing
biodegradable microspheres (5). Different types of shaking
and rotating devices have also been widely used for this
purpose (6). Franz diffusion cells, typically referred to as
“vertical diffusion cells” (VDC), were initially intended for
skin permeation. They were further modified to evaluate
nasal inserts and other mucosal dispersed systems (7–9).
Another attempt to develop in vitro tests for microspheres
was the elevated temperature accelerated assay, which
is useful mainly for batch-to-batch comparisons of long-
acting dosage forms, but they reflect neither the real-time
release rate nor the involved mechanism (3, 10, 11).
After the patent for the leuprolide-polylactic-co-glycolic
acid (LA–PLGA) delivery system was issued in the late
1980s, it was studied extensively (12, 13). The release
profile of the drug may be influenced by many parameters
such as physicochemical properties and drug loading,
variations of polymer molecular weight, lactic-to-glycolic
ratio, microencapsulation conditions, and in vitro test
protocols (14, 15). This particular system usually shows a
triphasic release profile characterized by an initial burst of
the drug near the surface or associated with pores after
polymer wetting, usually defined as the amount released
during the first 24 h (15), a lag phase until sufficient
polymer erosion has taken place, and a secondary burst
with approximately zero-order release kinetics (16–18).
This feature generally applies for all cases, but polymer
molecular weight, glass-transition temperature (Tg), drug
properties, and even device geometry play important
roles in precisely defining the release mechanism of a
given delivery system. When a low molecular weight PLGA
polymer is employed, for instance, the release is ruled
mostly by diffusion. Zolnik and Burgess (19) explained that
PLGA degrades from inside to outside at physiological pH.
Degradation begins with water going inward; hydrolysis
leads to the production of acidic oligomers, which are
retained within the microspheres because of the relative
hydrophobicity of the polymer, and the phenomenon
finally influences the degradation mechanism. When PLGA *Corresponding author.
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Dissolution Technologies | MAY 2012 7
is surrounded by a low pH medium, it becomes prone to
autocatalysis, which is also determined by the size and
porosity of PLGA microspheres. Moreover, tissue reaction
at the site where parenteral biopolymer-based delivery
systems are injected has to be considered (20). In vitro
assays should be as predictive as possible of physiological
events to achieve an accurate IVIVC. Still, controversy even
surrounds the selection of media that resemble in vivo
conditions most precisely. Although it has been highly
recommended to rely on USP apparatus already proven
for robustness, such as the USP Apparatus 4 equipment
already mentioned (21, 22), the quest for new suitable
easy-to-perform in vitro techniques still continues.
The aim of this work is to propose a reliable in vitro
procedure that mimics in vivo conditions for the
assessment of LA release from PLGA microspheres. Two
methods based on vertical diffusion cells and rotating
bottle apparatus have been tested to determine the in
vitro release of a water-soluble model drug from
polymeric microspheres. Both systems were modified with
dialysis membranes that separated the microspheres from
the external release medium, allowing the migration of
the drug without restriction. As leuprolide solubility in
water is very high, no violation of sink conditions was
suspected in any of the proposed methods. Both methods
were studied in different conditions and accompanied by
stability tests of leuprolide in the chosen media.
MATERIALS AND METHODS
Leuprolide acetate was obtained from Bachem
(Bubendorf, Switzerland). Poly(D,L-lactide-co-glycolide)
(PLGA) was Resomer RG502H (50% D,L-lactide and 50%
glycolide) with an inherent viscosity of 0.16–0.24 dL/g and
7,000–17,000 MW range (Boehringer Ingelheim, Ingelheim
am Rhein, Germany). Gelatin, polyvinyl alcohol (PVA),
methylene chloride, dimethylsulfoxide, Tween 80,
phosphate and sodium salts, and all other reagents were
of analytical quality.
Preparation and Characterization of Microspheres
PLGA microspheres containing LA were prepared by a
w/o/w emulsion. PLGA (8.0 g) was dissolved in 11 mL of
methylene chloride. Leuprolide acetate (0.907 g) was
added to a mixture of gelatin (0.16 g) and water (1 mL),
previously prepared. A w/o emulsion was formed by
mixing the aqueous leuprolide solution into the organic
phase using a homogenizer DIAX 900 (Heidolph,
Schwabach, Germany) at 22,000 rpm for 10 min in 30-sec
intervals in an ice water bath. The w/o emulsion was
added to a 1% PVA solution (400 mL) and homogenized at
22,000 rpm for 10 min. The obtained w/o/w emulsion was
stirred at 2,000 rpm for 1 h at 25 °C to allow the methylene
chloride to evaporate. After evaporation of the organic
solvent, the resulting microspheres were filtered through
a 100-µm pore membrane, washed three times by
centrifugation, and freeze-dried. Microspheres were stored
at 4 °C until needed.
Particle size and morphology were analyzed by
scanning electron microscopy (SEM Phillips 505,
Amsterdam, Holland). Samples were gold-sputtered with
an Edwards Sputter Coater S150B (Crawley, England).
Drug loading was determined by dissolution of PLGA
microspheres followed by HPLC/UV/FLUO. Briefly, 10 mg
of loaded microspheres was added to 10 mL of dimethyl-
sulfoxide and stirred until completely dissolved. The
chromatographic system consisted of a Waters 2695
Separations Module, Waters 996 DAD detector set at
228 nm, and Waters 2475 Multiwavelength Fluorescence
detector (Milford, Massachusetts, USA) set at excitation
and emission wavelengths of 280 and 325 nm,
respectively. The chromatographic column was a
C18 Synergi 4-µm Hydro-RP Phenomenex column,
150 × 4.6 mm (Torrance, California, USA), and the mobile
phase was methanol–0.25 M ammonium acetate (60:40)
at a flow rate of 0.8 mL/min. Drug loading was expressed
as mg LA/100 mg microspheres.
Stability Test of Leuprolide Acetate Solutions
LA was dissolved in PBS or acetate buffer at two concen-
tration levels, a low concentration level (0.01–0.02 mg/mL)
simulating the initial concentration (time zero) of LA in the
external medium of the in vitro release assay, and a high
concentration level (0.1–0.2 mg/mL) mimicking the final
cumulative amount of released LA. PLGA was added to
some of the specimens at a constant PLGA–LA ratio,
similar to that found in the microspheres. The flasks were
appropriately sealed to avoid evaporation and incubated
in an oven at 37 ± 0.5 °C for 31 days. The pH was measured,
and aliquots were withdrawn at fixed times and analyzed
by HPLC. Samples were vortexed for 1 min at the beginning
of the test and before each aliquot withdrawal.
In Vitro Release Test with Vertical Diffusion Cells
A Franz cell system (Hanson model 57–6M Manual
StartUp diffusion cell test system, Chatsworth, California,
USA) was built as follows. A dialysis membrane (molecular
cutoff 12,400 Da, Arthur Thomas Co., Philadelphia,
Pennsylvania, USA) was placed on the upper donor
chamber of the diffusion cell, separating this compart-
ment from the receptor chamber. An accurately weighed
quantity of LA-loaded microspheres (10 mg) was placed
on the membrane using a slab with an area of 1.77 cm2
and thickness of 1.2 mm. One milliliter of pH 7.4 150 mM
PBS buffer containing 0.1% w/v sodium azide and 0.05%
w/v Tween 80 (PBS-T buffer) to prevent microsphere
contamination and agglomeration, respectively, was
added on the membrane. In another set of experiments,
acetate buffer (pH 4.0) with 0.05% w/v Tween 80 was
used (acetate-T buffer). The receptor chamber was
completely filled with the corresponding buffer, wetting
the membrane and the microspheres. The acrylic top plate
was tightly sealed to avoid evaporation. At fixed intervals,
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Dissolution Technologies | MAY 20128
aliquots were withdrawn from some cells and replenished
with freshly prepared buffer. In some series of experiments
with PBS, the volume of the external medium was com-
pletely removed and replenished with freshly prepared
buffer. Tests were performed in triplicate at 37 ± 0.5 °C and
500 rpm. Samples were analyzed using the previously
described HPLC/UV/FLUO conditions. The cumulative
percentage released was calculated, and the mean values
and standard deviations were reported.
In Vitro Release Test with Rotating Bottle Apparatus
A rotating bottle apparatus (Alycar, Buenos Aires,
Argentina) that met NF XIV specifications was adapted for
slow rate control. Bottles were clipped into a rotating shaft
moving at 3 rpm that was immersed in a water bath at
37 ± 0.5 °C. Ten milligrams of accurately weighed LA
loaded microspheres was filled into a dialysis tube (flat
width: 2.54 cm, length: 10 cm, molecular cutoff: 12,400 Da)
embedded in 3.0 mL PBS-T buffer. Both ends of the dialysis
tubes were fastened with plastic seals. The dialysis bags
were placed inside glass test tubes containing 77.0 mL of
PBS-T buffer. The neck of each tube was sealed with
silicone to prevent any liquid exchange with the external
medium. Two-milliliter samples were withdrawn at
specified intervals from some tubes, and from others, the
complete volume was removed. In both cases, freshly
prepared buffer was used to replenish. Tests were
performed in triplicate, and samples were analyzed by
RESULTS AND DISCUSSION
Stability of Leuprolide Acetate in Different Media
To study LA stability in different release media as a
function of time, the drug was tested at two concentration
levels in two buffer solutions, PBS and acetate buffer. In
this way, the entire range of LA concentrations expected in
an in vitro release assay was considered. The pH was also
monitored in the bulk medium to relate pH changes to
LA stability. Although the change in pH was controlled by
buffer capacity (Table 1), the stability of the drug depend-
ed on the release medium. In PBS, LA was stable for at least
30 days at the highest concentration studied (Figure 1). A
10% LA loss was observed throughout time upon dilution
or PLGA addition. As shown in Table 1, only PLGA addition
at the highest level produced a pH decrease of 1.4 units.
On the other hand, no pH variations were observed in any
of the samples under acidic conditions. However, LA
proved to be unstable in acetate buffer, especially in the
presence of PLGA and at low concentrations (Figure 2).
LA incubated at acidic pH may suffer different degradation
pathways according to temperature and relative LA
concentration, as already reported (23). Yet, LA release
Table 1. pH Variation During LA Incubation at 37 °C in pH 4.0
Acetate and pH 7.4 Phosphate Buffers with and without PLGA
at Two Concentrations
Day 7 Day 14 Day 21 Day 31
LA low 7.3 7.3 7.3 7.3
LA high 7.3 7.2 7.3 7.3
PLGA 6.8 6.5 6.2 6.0
LA+PLGA low 7.3 7.2 7.2 7.2
LA+PLGA high 6.8 6.5 6.3 6.1
LA low 4.2 4.2 4.2 4.2
LA high 4.2 4.2 4.2 4.2
PLGA 4.2 4.2 4.1 4.0
LA+PLGA low 4.2 4.2 4.2 4.2
LA+PLGA high 4.2 4.2 4.1 4.0
Figure 1. Stability of LA during incubation at 37 °C in pH 7.4 PBS. Results are
expressed as percentage of initial concentration (n = 3).
Figure 2. Stability of LA during incubation at 37 °C in pH 4.0 acetate buffer.
Results are expressed as percentage of initial concentration (n = 3).
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Dissolution Technologies | MAY 2012 9
assays performed in pH 4.0 acetate buffer have been
reported (24), probably in an attempt to mimic the acidic
environment inside PLGA microspheres, as will be further
In Vitro Release Testing: Vertical Diffusion Cells and
Rotating Bottle Apparatus
Release profiles obtained with vertical diffusion cells
were clearly different depending on the release medium,
phosphate or acetate, but no differences were found
whether all of the external medium was replaced or an
aliquot was withdrawn (Figure 3), which confirms that
sink conditions had been maintained. The cumulative
percentage released in PBS buffer was 105.5 ± 4.3% for
aliquot removal and 101.2 ± 3.4% for complete volume
replacement. A triphasic release profile was observed
upon incubation in PBS buffer with 50% of the drug
released in the first 24 h. This kind of triphasic profile has
been described previously for PLGA microspheres using
different devices (3, 17, 18). In this case, it is hard to assume
that the high burst observed is only due to superficial,
nonencapsulated LA release. PLGAs of different molecular
weights are naturally glassy; their Tg values vary according
to their physicochemical structures above physiological
temperature. In a rubbery state, the mobility of polymer
chains and drug molecules increases, resulting in higher
drug release rates. An LA cumulative release of 86% after
30 h at 50 °C from microspheres made of 8,600 MW PLGA
(50:50) with a Tg of 40.04 °C has been reported (10). Even
though our tests were performed at 37 °C, we speculate
that a glassy–rubbery transition of the low molecular
weight fraction of the polymer (7,000–17,000 Da) might
have enhanced drug diffusion through the PLGA matrix
increasing the burst effect. The short lag phase commonly
assigned to erosion of the polymeric matrix can be
explained by the low molecular weight of the PLGA used
to prepare the microspheres together with the high water
solubility of the encapsulated peptide. In general, PLGA
release devices are considered bulk-erosion delivery forms
(25). However, it is assumed that in microspheres made of
a low molecular weight polymer, the phenomenon that
rules drug release is diffusion. Surface pores and cavities
filled with medium in the microsphere matrix are path-
ways for molecules, both monomer and drug. In our case,
diffusion was the key point that defined release rates and
profiles. The use of a system with a low molecular weight
polymer and the developed concentration gradient aided
rapid drug diffusion. The high final cumulative percentage
confirms drug stability throughout the assay. This can be
explained by a fluid access of buffer to the easily eroded
polymeric system, which prevents the formation of a low
pH environment that would affect drug stability as
On the other hand, a maximum of 30.4 ± 1.8% was
released after 25 days in acetate buffer, afterward decreas-
ing to a cumulative 15.2 ± 1.8% by the end of the assay
(Figure 4). In this case, no burst release was obtained;
instead, a lag time was observed in the first week, followed
by a first-order release and a post-maximum decrease. The
generation of an acidic environment inside PLGA micro-
spheres has been studied extensively in vitro with pH-
sensitive probes (26, 27). The degradation of the polymer
promotes pH decrease, which varies from very low
(approximately pH 1.5) at the center of the microsphere
to higher values at its boundaries. Our results show that
LA is unstable in acetate buffer at low concentrations
and especially in the presence of PLGA (Table 1), which is
in accordance with the corresponding release profile
(Figure 4). In fact, although buffer seems to regulate the
medium pH, LA degradation at pH 4 is most probably the
cause of the low final cumulative percentage released and
Figure 3. Cumulative in vitro release of leuprolide from PLGA microspheres
using Franz diffusion cells and pH 7.4 PBS at 37 °C; (●) aliquots removed at
fixed intervals, (□) the complete volume of the external medium removed
(n = 3).
Figure 4. Cumulative in vitro release of leuprolide from PLGA microspheres
using Franz diffusion cells and pH 4.0 acetate buffer at 37 °C (n = 2).
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Dissolution Technologies | MAY 201210
its subsequent decrease. As previously mentioned, no LA
degradation was found in pH 7.4 PBS buffer, either in the
stability test or in the in vitro release test, which led us to
choose this neutral isotonic buffer for further assays.
Release profiles obtained with the rotating bottle
apparatus were markedly different from those obtained
with VDC. Though the cumulative percentage released
was 110.1 ± 20.5%, the pattern did not fit a triphasic
model (Figure 5). A high percentage of the drug was
rapidly released in the burst phase, and complete release
was achieved early during the test. High variability among
individual values was also observed throughout the assay.
Similar results were obtained either by removing aliquots
or by complete volume replacement (data not shown). The
high surface area available for multidirectional diffusion
and the slow but constant movement may have promoted
an accelerated drug migration, while the high dilution
increased the variability of data.
Although erosion and swelling may occur according to
the characteristics of each polymer, diffusion is always
involved in the release mechanism. The rotating bottle
apparatus promotes diffusive release, and the effect of LA
high water solubility can be magnified in this device. In
fact, microspheres move freely inside the dialysis sac at a
very low speed, which prevents aggregation. In VDC,
though microspheres do not move, they are placed in the
buffered donor compartment in contact with a smooth
surface, contrary to magnetically agitated dialysis bags (5).
In this case, the release is actually a two-step process, the
diffusion of LA through the polymer matrix and its
subsequent diffusion from donor to receptor chamber.
Therefore, diffusion cells may resemble in vivo behavior
more closely, as microspheres are confined to an area but
fully imbibed in release medium. When tested for a
mucosal delivery system, this device simulated well the
quantities of water on mucosal surfaces in vivo (9). As
described by Klose et al. (28), when the drug is released
from the microsphere, it encounters tissue instead of a
medium without hindrance. To mimic what happens in
vivo, there should be a balance between totally dispersed
particles and lump-like aggregation. In our opinion, Franz
diffusion cells seem to fulfill this requirement.
Franz diffusion cells and a rotating bottle apparatus
were tested in an attempt to find an in vitro release
method that may mimic the in vivo behavior of
biopolymer-based delivery systems for a water-soluble
drug. Leuprolide acetate, which is stable at physiological
conditions even in the presence of PLGA, displayed
triphasic and biphasic release profiles in Franz diffusion
cells and the rotating bottle apparatus, respectively. VDC,
where the microspheres are partially confined but sink
conditions are still maintained, appeared to be a suitable
alternative to the existing USP Apparatus 4. Further
investigations will continue with different biodegradable
polymers tested under similar conditions and contrasted
to in vivo profiles to establish if Franz diffusion cells can be
considered as an alternative in vitro method for predicting
in vivo behavior of prolonged-release biodegradable
The authors would like to thank Dr. Nora François and
Dr. Marta Daraio for technical assistance in the calcula-
tions. This work was supported by PTA grant 125/08 from
the National Institute of Industrial Technology, Argentina.
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