Photoacoustic methods for in vitro study of kinetics progesterone release

from the biodegradation of polyhydroxybutyrate/polycaprolactone used as

intravaginal devices
N. E. Souza Filho, V. V. G. Mariucci, G. S. Dias, W. Szpak, P. H. P. Miguez et al.
Citation: Appl. Phys. Lett. 103, 144104 (2013); doi: 10.1063/1.4823986
View online: http://dx.doi.org/10.1063/1.4823986
View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v103/i14
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APPLIED PHYSICS LETTERS 103, 144104 (2013)

Photoacoustic methods for in vitro study of kinetics progesterone release

from the biodegradation of polyhydroxybutyrate/polycaprolactone used as
intravaginal devices
N. E. Souza Filho,1,2,a) V. V. G. Mariucci,1 G. S. Dias,1 W. Szpak,1 P. H. P. Miguez,3
E. H. Madureira,3 A. N. Medina,1 M. L. Baesso,1 and A. C. Bento1
1

Universidade Estadual de Maring

a, Departamento de Fsica, Grupo de Estudos dos Fen^
omenos
Fotot
ermicos-GEFF/DFI/UEM, Av. Colombo 5790, Maring
a-PR, Brazil
2
Universidade Federal de Santa Maria, Departamento de Eng. Ac
ustica, Av. Roraima 1000, CEP 97105900,
Santa Maria-RS, Brazil
3
Universidade de S~
ao Paulo, Departamento de Reproduc~
ao AnimalVRA/USP, Av. Prof. Dr. Orlando
Marques Paiva 87, S~
ao PauloSP, Brazil

(Received 8 June 2013; accepted 20 August 2013; published online 1 October 2013)
Intravaginal devices composed of polyhydroxybutyrate/polycaprolactone blends incorporating
progesterone were used over eight days in crossbred cow ovariectomized, and then analyzed with
photoacoustic methods, measuring the absorption spectra, thermal diffusivity, and inspecting its
degradation by means of scanning electron microscopy. The characteristic time found for
progesterone release was TR  53 h, and the typical time found for biodegradation was TB  30 h.
Morphological analysis complements the study showing that release of progesterone and
C 2013 AIP Publishing LLC.
biodegradation of the blend occurs on sample surface. V
[http://dx.doi.org/10.1063/1.4823986]
The artificial insemination is essential in livestock to be
a biotechnical that provides the genetic improvement of cattle. One of the protocols makes use of intravaginal devices
for progesterone releasing. Most systems delivery of veterinary drugs is based on silicone, polyurethane, vinyl acetate,
or ethylene, which are cheap and biocompatible.13
The biodegradable products have various applications,
with significant advances in the biomedical implant and drug
delivery system. For animal health care, for example, often
the duration of drug release should be extended.1 In this context, some positive results have been obtained with drug
incorporation in biopolymers, which permits a better release
control of the drug from the biodegradation of the
material.46
One of physiological effects of progesterone is to inhibit
the growth of epithelial cells of human breast.7 Progesterone
produces its effect on target cells by enhancing the synthesis
of new structural proteins or enzymes.8 Among the steroids,
progesterone is the most attractive to regulate fertility and
treat infertility. The P4 occurs in high concentrations in the
body and has no toxicity problems, as usually happens with
synthetic progestins. However, progesterone is not active
orally, except in high doses, and have a relatively biological
short lifetime. In an attempt to reduce production costs and
environmental impacts, Nascimento et al.9 developed intravaginal devices for sustained release of progesterone composed by polyhydroxybutyrate (PHB) and polycaprolactone
(PCL) containing amount of P4.9
This paper reports the study of parenteral delivery of
P4 by biodegradation of the intravaginal device by

a)

Authors to whom correspondence should be addressed. Electronic

addresses: nilson.evilasio@eac.ufsm.br and acbento@uem.br. Tel./Fax:
55-44-3026 4623.

0003-6951/2013/103(14)/144104/4/$30.00

photoacoustic techniques. Samples of the used devices were

submitted to spectroscopic analysis using photoacoustic
spectroscopy (PAS), thermal diffusivity with open photoacoustic cell (OPC), and degradation by means of scanning
electron microscopy (SEM), all aiming to understand how
the hormone penetrates and spreads through the biopolymer.
To observe the total release of progesterone in vivo during the eight days it was held the following: On day 0 (D0),
before the insertions of the devices in each cow, it was collected a sample to obtain the total amount of P4 for each device. The D0 devices were inserted on a herd, then removed
every 24 h to obtain individual samples, and inserted into the
cows again. Usually, from D0 to D8 one obtain the net
amount for each device when subtracting the measured value
of the P4 taken for each day with respect to sample that was
collected at D0. The bovine intravaginal device ProgestarV
(Inovare, Brazil) was produced in Rheology and Processing
Center in the Department of Materials Engineering (DEMA UFSCAR). The device has a proportion of 46:46:8 of PHB,
PCL, and P4, respectively. The experiments were performed
two times to obtain the total release of progesterone over
eight days. It was used six crossbred cows, Bos taurus
crossed with Bos taurus indicus ovariectomized, from the
Department of Animal Reproduction (VRA), Faculty of
Veterinary Medicine (FMVZ), University of S~ao Paulo.
After retrieved from cows, several samples were cut in
disks with a diameter of 5 mm with different thicknesses and
named as Dj (j number of days) according time passing in
days after implantation. The sample D0 means the before the
introduction of the intravaginal device [(PHB/PCL) P4],
and the sample D8 means the eight days after the device
implantation.
The samples presented a large absorption band in the
UV region and were measured in a photoacoustic conventional apparatus (closed photoacoustic cell), whereas thermal

103, 144104-1

C 2013 AIP Publishing LLC

V

144104-2

Filho et al.

diffusivities were measured using the OPC technique. The

sample is disposed on the top of the microphone, sealing its
aperture, and the other sample side stays exposed to a modulated light beam, which generates the thermal excitation.10 A
solid-state laser (Laser Aperture BWT-50-E) with 45 mW of
power and wavelength of 532 nm was used.
The OPC theory predicts two limiting cases for an opaque sample, relating the thickness (ls) and its thermal diffusion length ls a=pf 1=2 , where as is the samples thermal
diffusivity and f is the optical modulation frequency.
Therefore, in short the amplitude of OPC S(f) follows:10
(1) For a thermally thin (ls  ls), where the amplitude of
the microphone signal (S) is attenuated with increasing
frequency (f ) such that Sf / f 3=2 .
(2) For thermally thick (ls  ls), where the amplitude of
the microphone signal (S) is attenuated with p
increasing

frequency (f ) such that, Sf / 1=f expb f .

Thermal diffusivity is obtained from best fit of b parameter, which gives thermal diffusivity a p2s =b2 .
Morphology of the biopolymers matrix and the pure hormone (P4 99%) were performed with SEM analysis in gold
coated using a Shimadzu SuperScan SS-500.
Figure 1(a) shows the photoacoustic absorption spectra
for pure progesterone (P4 99%); sample D0; sample D0 with
sanded surface; and the PHB/PCL-base blend (PHB 50%
and PCL 50%, without P4). The PAS spectra show a peak
around 240 nm, observed in all spectra and attributed to the
PHB/PCL blend. The characteristic absorption band of the
hormone was identified to be on range of 270380 nm. Since
the sample D0 has a much lower concentration of P4 in its
composition, compared with pure P4 sample, the characteristic absorption curve of the P4 powder was normalized to be
compared under the same scale to the doped samples, where
the typical hormone band is well defined. A removal of a
thin surface layer of D0 (named D0-sanded) was enough to
decrease the characteristic band of the hormone, which it
becomes much like the absorption of the sample PHB/PCLbase (Fig. 1(a)). Figure 1(b) shows the PAS spectra just for a

Appl. Phys. Lett. 103, 144104 (2013)

few samples for a better view: D0; D1; D3; D5; D7; D8 and
the PHB/PCL-base sample, where one can see a gradual
decrease in the absorption band of the hormone P4 and its
relation with time of insertion. One may note that sample D8
tends to the blend PHB/PCL-base but still presents remains
of P4 in the range 270300 nm.
The typical exponential decay of OPC amplitude after
linearization undergoes to best fit what gives parameter b,
corresponding to the individual slope (Dj) related to time
releasing, slopes were obtained from 36 to 100 Hz in a semilog plot.
Table I summarizes the main results, giving the samples
thicknesses, the fitted parameters b and its confidence band,
also the calculated value for thermal diffusivity a of each
PHB/PCL blend. Errors for a is calculated from single fittings of b and comprises both, thickness error dL  1 lm
(1/5000.2%) and db  0.006 (0.006/0.9000.7%). One
can evaluate the error da from derivative of a p2s =b2 ,
which gives da 2adb=b d= and one can estimate
it close to 2%. Nevertheless, a major error is found from fitting b using more than one single sample. Then one can estimate statistically an averaged value from measuring one
sample at a time of a triplicate set in such way errors could
be overestimated reaching higher values like those in Table I
for a, figuring in percentage about (da/a)  7.4%.
Figure 2(a) shows the in time variation to the integrated
areas of the curves like those presented in the Figs. 1(a) and
1(b). The band 270380 nm was used in the integration of
hormone absorption areas. The time to decrease the amount
of P4 in the sample to a value of 1/e of the sample D0 was
denominated as the characteristic time of release (TR). The
fitted data to an exponential decay revealed a TR  53 h,
which is the best adjusted time for the releasing process, in
which 63% of the total amount of P4 is released when device
is intravaginally implanted.
Time evolution of thermal diffusivity of PHB PCLblends is shown in Fig. 2(b) for each sample Dj. The main
picture is the exponentially growing of thermal diffusivity
along the time of intravaginal insertion. A rapid releasing of
P4 is found at about two days and a slow growing till the
thermal diffusivity finding a plateau and a steady value
around to a8  6.5  103 cm2/s. The typical time needed for
the diffusivity grow over 63% of its steady value (sample
D8) is evaluated as the typical time of biodegradation of
TABLE I. Calculated thermal diffusivity of P4-doped blend of PHB/PCL
using blend thicknesses and slopes of OPC fittings.

Blend

FIG. 1. (a) Photoacoustic comparative for optical absorption spectra

between pure powder of P4 (99%) against charged blend D0, blend D0
sanded, and PHB PCL-base without P4; (b) photoacoustic absorption
spectra for some blend samples, charged and prior implantation D0, and after retrieved from cows D1, D3, D5, D7, and D8. PHB PCL-base is plotted
to show the threshold of P4 release approaching to the blend base without
hormone.

D0
D1
D2
D3
D4
D5
D6
D7
D8
PHB PCL-base

Thickness
ls (104 cm)

Parameter:
b (s1/2)

Thermal diffusivity
a (103 cm2/s)

405 6 1
454 6 1
420 6 1
418 6 1
413 6 1
503 6 1
409 6 1
395 6 1
410 6 1
415 6 1

(0.922 6 0.007)
(1.006 6 0.007)
(0.925 6 0.005)
(0.922 6 0.005)
(0.907 6 0.005)
(1.101 6 0.006)
(0.892 6 0.007)
(0.860 6 0.008)
(0.897 6 0.006)
(1.011 6 0.007)

6.06 6 0.45
6.39 6 0.47
6.48 6 0.48
6.45 6 0.48
6.51 6 0.48
6.55 6 0.49
6.60 6 0.49
6.62 6 0.49
6.56 6 0.49
5.29 6 0.46

144104-3

Filho et al.

Appl. Phys. Lett. 103, 144104 (2013)

PHB PCL-blends. Fitting data in Fig. 2(b) to an associated

exponential at as 1  expt=TB the biodegradation
typical time is found TB  30 h, which is interpreted as the
characteristic time for blend surface to degrade after being
inserted.
Figure 3 shows SEM micrograph results for progesterone standard P4 (Fig. 3(a)), pure blend surface of
PHB PCL-blend (Fig. 3(b)), sample D0 surface before the
introduction of the intravaginal device (Fig. 3(c)), and the
cross section sample D0 (Fig. 3(d)). The micrograph shown
in Fig. 3(a) shows a clear difference between P4 and the texture of polymeric matrix of PHB PCL-base (Fig. 3(b)).
The picture shows that the hormone pure (P4 99%) is predominantly spherical symmetry with variations in diameter
from 1.25 to 5.00 lm. In contrast, PHB PCL-base presents
a completely smooth surface, with soft undulations and some
microcracks. Furthermore, Fig. 3(c) shows final texture
result after adding the P4 hormone to sample D0. Figure
shows that sample D0 is seen as a mixture of composed
blocks in rhombic compact form with size ranging 520 lm,
which one can find it similar to granules of amide. The cross
section micrograph of the sample D0 showed in Fig. 3(d)

indicates that granular characteristic occurs only on the surface and it extends up to a 20 lm depth approximately.
The ultimate characteristic found from SEM analysis if
depicted in Fig. 4, where one can find an evident block size
reduction when it is compared Fig. 4(a) against Fig. 3(c) that
shows sample D0 surface. Moreover, it is worthy to note the
reduction of block size from approximately 20 lm5 lm, if
one compares sample D1 (Fig. 4(a)) to D8 (Fig. 4(c)). The
cross-section of samples D1 and D8 shown in Figs. 4(b) and
4(d) indicates a typical depth of 20 lm where degradation
and consequently progesterone releasing may be found.
Here, a polymer and the active agent have been mixed to
form a homogeneous system, also referred to as a matrix system (PHB PCL-blend). Mass diffusion occurs when the
drug (progesterone P4) passes from the polymer matrix
(PHB PCL-blend) into the external environment (intravaginal medium). As the hormone release continues, its rate normally decreases with this type of system, since the active
agent has a progressively longer distance to travel and therefore requires a longer diffusion time to release. Thus, aside
releasing the drug, degradation also may occur and SEM
micrograph looks to be well correlated with microstructure
of the blend and to thermal diffusivity evolution with time.
There are some known theoretical models of drug
release in the literature,1113 but the case of the drug delivery
followed degradation has been studied recently in a device
silk-based for oral administration.8 It is presented here an alternative way to relate the kinetics of progesterone release
in vivo using the integrated areas of the PAS spectra and
measuring the evolution of thermal diffusivity in time.
In reservoir systems, the release rate is well established,
and it is independent on environment, with advantage of
keeping constant the concentration of drug inside the host
body.3 The drug delivery from bulk-eroding or surfaceeroding biodegradable or layer-by-layer14 systems are named
of Chemically Controlled Systems, the drug may be uniformly distributed and immobilized in the matrix, and the
release occurs by erosion of the matrix of the biomaterial.
When there are covalent bonds between the drug and the biopolymer, the release will occur through the divisions of the
connections formed through chemical reactions, usually enzymatic or hydrolytic degradation of the matrix.3

FIG. 3. Micrograph comparative for samples in Fig. 1. (a) Progesterone P4

standard with 99% (2000); (b) biopolymer blend [PCL PHB] with no P4
charge (2000); (c) surface of blend D0 [PHB PCL P4] full charged
before implantation (500); and (d) cross section of blend D0 detailing P4
partially in the surface (500).

FIG. 4. Micrograph comparative two samples of Fig. 2 after retrieved from

cow. (a) Surface of sample D1 (period lasts 1 day) (500); (b) cross section
of sample D1 (500); (c) surface of sample D8 (period lasts 8 day) (500);
(d) cross section of sample D8 (500).

FIG. 2. (a) Integrated area of photoacoustic absorption of Fig. 2 in the range

200400 nm. The evolution in time shows that area decreases exponentially
with period of intravaginal P4 release; (b) results from the fittings for b.
Thermal diffusivity evolution with period of intravaginal P4 release shows
fast increasing up to 2 days followed by a steady value after about 8 days of
P4 release.

144104-4

Filho et al.

Most biodegradable polymers are designed to degrade

as a result of hydrolysis of the polymer chains into biologically acceptable, and progressively smaller, compounds.
Degradation may take place through bulk hydrolysis, in
which the polymer degrades in a fairly uniform manner
throughout the matrix. For some degradable polymers, most
notably the polyanhydrides and polyorthoesters, the degradation occurs only at the surface of the polymer, resulting in a
release rate that is proportional to the surface area of the
drug delivery system. The drug can be dissolved, dispersed,
or partially dissolved in the polymer matrix. The release of
drug from stable polymers is based on diffusion, which can
occur as either zero or first order kinetics. The release of
drug from biodegradable polymers is predominantly a consequence of diffusion of drug molecule and simultaneous degradation of polymer matrix. In addition, there are multiple
other factors that need to be taken into account when drug
releasing devices are being developed.12
The rate of release can be controlled by the geometry of the
biopolymer,13 for many applications the geometry of the
implant may be limited. The external environmental properties
such as pH, temperature, and enzymes15 can also affect the
kinetics of drug release and polymer degradation in vivo. This is
particularly the case of natural biomaterials like silk, collagen,5
and PHB or PCL, which are subject to enzymatic degradation.
In describing drug release from such systems, it is necessary to consider all possibilities that can affect the release of
drug, which makes the accurate determination of release
kinetics very complicated and making it necessary to use alternative techniques to estimate the release. In Figs. 4(c) and
4(d) there is a porous structure with granular region reduction
from approximately 5 lm, suggesting the degradation and removal of the surface layer. Degradation occurs by hydrolysis
due to surface contact with the mucosa. Some characteristics
such as roughness, fracture, fragmentation, changes color or
formation of biofilms on the surface of the polymer may be
indications of microbial attack. The surface changing, due to
microbial action in the biopolymer, enhances colonization by
microorganisms and even exposes the polymer into contact
with enzymes that act on the carbons of the carbonyl groups,
the sites of attack characteristic of these enzymes.14
The biodegradation occurs initially in the amorphous
regions of the polymeric blend which is in contact with the
mucosa and hence the contact surface of device becomes
scaly. The scale does not have directional order due to skin
friction (a parasitic drag component by cervical mucus) and
their dimensions ranging from 2 to 5 lm, as SEM shows in
Figs. 4(b) and 4(d). The macromolecule is reacted with water
and hydrolyzed into smaller fragments that can be used as
nutrients for microorganisms. The amorphous parties are characterized by an arrangement of molecules disordered, irregular in shape, and has no structural order in their chains. The
micrograph of the cross section of the sample D1 (Fig. 4(b))
and D0 (Fig. 3(d)) confirms that the material is predominantly
homogeneous and that biodegradation takes place only in the
surface of contact. Progesterone is gradually released as a
function of time in the body of the animal via diffusion, after
removal of the surface layer by biodegradation.

Appl. Phys. Lett. 103, 144104 (2013)

Although the micrographs reveal the information that

the biodegradation of the blend and the release of P4 are in
the surface, it remains difficult to estimate a biodegradation
rate or release kinetics. In this sense, both PAS spectroscopy and OPC methods are shown to be good alternatives
to overcome the difficulties in determining typical time of
biodegradation and release. The evolution of the micrographs in Fig. 4 indicates that the attack of microorganisms
occurs primarily in the amorphous parts of the blend surface and the degree of this degradation reflects in the
changes of thermal diffusivity as monitored by OPC measurements. The data were correlated with typical time of biodegradation of blend TB. In the case of intravaginal devices
the degradation is slow (TR  53 h) and diffusion of P4
occurs faster (TB  30 h) reflecting degradation effect on
the bulk of blends.
In conclusion, these typical times of biodegradation and
releasing of P4 occurs correspondently to the follicular phase
of the bovine estrous cycle, which occurs in a high production of estrogen E4 and low production of P4. The PHB/PCL
blend is a good substitute for silicon in intravaginal devices.
The progesterone delivery followed by the degradation of
the blend in vivo must take into account several factors,
which makes it difficult the exact determination of release
kinetics. As an alternative to overcome these difficulties, one
may use photoacoustic techniques. Finally, results from PAS
and OPC have shown to be consistent in terms of typical
characteristic time release and well correlated to biodegradation of the carrier device based on blends of PHB PCL.
The authors acknowledge the Brazilian agency CAPES,
CNPQ, FAPESP, and Fundac~ao Araucaria for partial financial support.
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