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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 

Published by the AIP Publishing LLC. 

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Photoacoustic methods for in vitro study of kinetics progesterone releasefrom the biodegradation of polyhydroxybutyrate/polycaprolactone used asintravaginal 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

1Universidade Estadual de Maringa, Departamento de Fısica, Grupo de Estudos dos Fenomenos Fotot ermicos-GEFF/DFI/UEM, Av. Colombo 5790, Maringa-PR, Brazil2Universidade Federal de Santa Maria, Departamento de Eng. Acustica, Av. Roraima 1000, CEP 97105–900,Santa Maria-RS, Brazil3Universidade de S~ao Paulo, Departamento de Reproduc~ao Animal–VRA/USP, Av. Prof. Dr. Orlando

 Marques Paiva 87, S~ao Paulo–SP, 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 30h.

Morphological analysis complements the study showing that release of progesterone and

biodegradation of the blend occurs on sample surface. VC  2013 AIP Publishing LLC.

[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 cat-

tle. One of the protocols makes use of intravaginal devices

for progesterone releasing. Most systems delivery of veteri-

nary drugs is based on silicone, polyurethane, vinyl acetate,

or ethylene, which are cheap and biocompatible.1 – 3

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 con-

text, some positive results have been obtained with drugincorporation in biopolymers, which permits a better release

control   of    the drug from the biodegradation of the

material.4 – 6

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 andenvironmental impacts, Nascimento  et al.9 developed intra-

vaginal devices for sustained release of progesterone com-

posed 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

photoacoustic techniques. Samples of the used devices were

submitted to spectroscopic analysis using photoacoustic

spectroscopy (PAS), thermal diffusivity with open photoa-

coustic 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  dur-

ing the eight days it was held the following: On day 0 (D0),

before the insertions of the devices in each cow, it was col-

lected a sample to obtain the total amount of P4 for each de-

vice. The D0 devices were inserted on a herd, then removedevery 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 Progestar VR

(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 (j¼ number of days) according time passing in

days after implantation. The sample D0 means the before the

introduction of the intravaginal device [(PHB/PCL)þP4],

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 conven-

tional apparatus (closed photoacoustic cell), whereas thermal

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   VC  2013 AIP Publishing LLC103, 144104-1

APPLIED PHYSICS LETTERS 103, 144104 (2013)

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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 modu-

lated 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 opa-

que sample, relating the thickness (ls) and its thermal diffu-

sion length  ls ¼ ða=p f Þ1=2, where as  is the sample’s thermaldiffusivity 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 Sð f Þ /  f 3=2.

(2) For thermally thick (ls   ls), where the amplitude of 

the microphone signal (S) is attenuated with increasing

frequency ( f ) such that, Sð f Þ / ð1= f Þexpðb ffiffiffi 

 f p  Þ.

Thermal diffusivity is obtained from best fit of “b” pa-

rameter, which gives thermal diffusivity   a ¼ ðp‘2s=b2Þ.

Morphology of the biopolymers matrix and the pure hor-

mone (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 270–380 nm. Since

the sample D0 has a much lower concentration of P4 in its

composition, compared with pure P4 sample, the characteris-

tic absorption curve of the P4 powder was normalized to be

compared under the same scale to the doped samples, wherethe 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/PCL-

base (Fig. 1(a)). Figure 1(b) shows the PAS spectra just for a

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 270–300 nm.

The typical exponential decay of OPC amplitude after 

linearization undergoes to best fit what gives parameter b,

corresponding to the individual slope (D j) related to timereleasing, slopes were obtained from 36 to 100 Hz in a semi-

log 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 fit-

tings of   b   and comprises both, thickness error   dL 1 lm

(1/500–0.2%) and   db 0.006 (0.006/0.900–0.7%). One

can evaluate the error   da   from derivative of   a ¼ ðp‘2s=b2Þ,

which gives  da ¼ 2a½ðdb=bÞ þ ðd‘=‘Þ  and one can estimate

it close to 2%. Nevertheless, a major error is found from fit-

ting b using more than one single sample. Then one can esti-

mate 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 270–380 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 53h,

which is the best adjusted time for the releasing process, in

which 63% of the total amount of P4 is released when deviceis intravaginally implanted.

Time evolution of thermal diffusivity of PHBþPCL-

blends 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 

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

between pure powder of P4 (99%) against charged blend D0, blend D0

sanded, and PHBþPCL-base without P4; (b) photoacoustic absorption

spectra for some blend samples, charged and prior implantation D0, and af-

ter retrieved from cows D1, D3, D5, D7, and D8. PHBþPCL-base is plotted

to show the threshold of P4 release approaching to the blend base without

hormone.

TABLE I. Calculated thermal diffusivity of P4-doped blend of PHB/PCL

using blend thicknesses and slopes of OPC fittings.

Blend

Thickness

ls (10

4cm)

Parameter:

b (s1/2

)

Thermal diffusivity

a (103

cm2 /s)

D0 4056 1   (0.9226 0.007) 6.066 0.45

D1 4546 1   (1.0066 0.007) 6.396 0.47

D2 4206 1   (0.9256 0.005) 6.486 0.48

D3 4186 1   (0.9226 0.005) 6.456 0.48

D4 4136 1   (0.9076 0.005) 6.516 0.48

D5 5036 1   (1.1016 0.006) 6.556 0.49

D6 4096 1   (0.8926 0.007) 6.606 0.49

D7 3956 1   (0.8606 0.008) 6.626 0.49

D8 41061   (0.8976 0.006) 6.566 0.49

PHB

þPCL-base 41561

  (1.0116 0.007) 5.296 0.46

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PHBþ

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

exponential   aðt Þ ¼ as½1 expðt =TBÞ   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 progester-

one standard P4 (Fig.   3(a)), pure blend surface of 

PHBþPCL-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 tex-

ture of polymeric matrix of PHBþPCL-base (Fig.   3(b)).

The picture shows that the hormone pure (P4 99%) is pre-

dominantly spherical symmetry with variations in diameter from 1.25 to 5.00 lm. In contrast, PHBþPCL-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 5–20 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 sur-

face 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 lm–5 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) and4(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 sys-

tem (PHBþPCL-blend). Mass diffusion occurs when the

drug (progesterone P4) passes from the polymer matrix

(PHBþPCL-blend) into the external environment (intravag-

inal medium). As the hormone release continues, its rate nor-

mally decreases with this type of system, since the active

agent has a progressively longer distance to travel and there-

fore 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,11 – 13 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 al-

ternative 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 surface-eroding biodegradable or layer-by-layer 14 systems are named

of Chemically Controlled Systems, the drug may be uni-

formly 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 bio-

polymer, the release will occur through the divisions of the

connections formed through chemical reactions,  usually en-

zymatic or hydrolytic degradation of the matrix.3

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

200–400nm. 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.

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

standard with 99% (2000); (b) biopolymer blend [PCLþPHB] with no P4

charge (2000); (c) surface of blend D0 [PHBþPCLþP4] 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).

144104-3 Filho et al.   Appl. Phys. Lett. 103, 144104 (2013)

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Most biodegradable polymers are designed to degrade

as a result of hydrolysis of the polymer chains into biologi-

cally 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 degrada-

tion occurs only at the surface of the polymer, resulting in a

release rate that is proportional to the surface area of thedrug 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 conse-

quence of diffusion of drug molecule and simultaneous deg-

radation 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 neces-

sary 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 al-

ternative 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 re-

moval of the surface layer. Degradation occurs by hydrolysis

due to surface contact with the mucosa. Some characteristicssuch 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 inFigs. 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 char-

acterized by an arrangement of molecules disordered, irregu-

lar 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.

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 spectros-

copy 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 micro-

graphs in Fig. 4  indicates that the attack of microorganisms

occurs primarily in the amorphous parts of the blend sur-face and the degree of this degradation reflects in the

changes of thermal diffusivity as monitored by OPC meas-

urements. The data were correlated with typical time of bio-

degradation 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 produc-

tion 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 biodegrada-

tion of the carrier device based on blends of PHB þPCL.

The authors acknowledge the Brazilian agency CAPES,

CNPQ, FAPESP, and Fundac~ao Araucaria for partial finan-

cial support.

1G. Winzenburg, C. Schmidt, S. Fuchs, and T. Kissel,  Adv. Drug Delivery

Rev.  56, 1453 (2004).2S. K. Panday, C. Haldar, D. K. Patel, and P. Maiti,  Advances in Polymer 

Science (Springer-Verlag, Berlin, 2013).3V. A. S. Vulcani, D. G. Macoris, and A. M. G. Plepis,  Cienc. Rural   38,

1329 (2008).4A. P. Bonartsev, G. A. Bonartseva, T. K. Makhina, V. L. Myshkina, E. S.

Luchinina, V. A. Livshits, A. P. Boskhomdzhiev, V. S. Markin, and A. L

Iordanskii, Appl. Biochem. Microbiol. 42, 625 (2006).5

E. M. Pritchard, T. Valentin, D. Boison, and D. L. Kaplan,   Biomaterials

32, 909 (2011).6S. Mehrotra, D. Lynam, R. Maloney, K. M Pawelec, M. H. Tuszynski, I.

Lee, C. Chan, and J. Sakamoto,  Adv. Funct. Mater.  20, 247 (2010).7J. Desreux, F. Kebers, A. Noel, D. Francart, H. Van Cauwenberge, V.

Heinen, J. L. Thomas, A. M. Bernard, J. Paris, R. Delansorne, and J. M.Foidart, Eur. J. Cancer  36, S90 (2000).

8G. Sager, A. Orbo, R. Jaeger, and C. Engstrom,  J. Steroid Biochem. Mol.

Biol. 84, 1 (2003).9

J. F. Nascimento and W. M. Pachekoski, U.S. patent 20090291119A1

(2009).10

A. C. Bento, D. T. Dias, L. Olenka, A. N. Medina, and M. L. Baesso,

Braz. J. Phys.  32, 483 (2002).11P. L. Ritger and N. A. Peppas,  J. Controlled Release 5, 37 (1987).12

T. Higuchi, J. Pharm. Sci.  52, 1145 (1963).13K. Kosmidis, E. Rinaki, P. Argyrakis, and P. Macheras,  Int. J. Pharm.  254,

183 (2003).14

G. Decher, Science 277, 1232 (1997).15

T. Yoshioka, N. Kawazoe, T. Tateishi, and G. Chen,   Biomaterials   29,

3438 (2008).

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