VETERINARY DRUG RELEASE BY NANOSTRUCTURED
POLYMERS
Juliano E. Oliveira1, Ed H. Madureira2, Luiz H.C. Mattoso3, Odílio B.G. Assis3
1
Depto. de Engenharia de Materiais, Universidade Federal da Paraíba, João Pessoa (PB), Brasil
2
Depto. de Reprodução Animal, Universidade de São Paulo, Pirassununga (SP), Brasil
3Lab. Nacional de Nanotecnologia para o Agronegócio, Embrapa Instrumentação, São Carlos (SP), Brasil
E-mail: juliano@ct.ufpb.br
Abstract. Several techniques are used to obtain polymeric nanofibers. Solution blow spinning (SBS) is a
technology feasible to produce nanostructured polymeric membranes loaded with active agents. In the
present study, nanofibrous mats of biopolymers like poly(lactic acid) (PLA) and zein loaded with
medroxyprogesterone (MP4) were produced by SBS at different P4 concentrations. The spun membranes
were characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC).
Interactions between progesterone and PLA were confirmed by rheological measurements of the
biopolymer/P4 solutions and in the spun mats by microscopy (SEM), and thermal (DSC) analyses. The in
vitro releasing of MP4 was evaluated using a high-performance liquid chromatography (HPLC). SEM
micrographs provided evidences of a smooth and homogeneous structure for nanostructured membranes
without progesterone crystals on fiber surface. Moreover, the presence of progesterone affects the fiber
morphology. X-Ray analysis indicated that size of PLA crystallites increased with progesterone content.
Finally, by in vitro release experiments it was possible to observe that the progesterone releasing in PLA
fibers follows nearly first-order kinetics, probably due to the diffusion of hormone into solid matrix.
Keywords: biopolymer, hormone, drug release, veterinary medicine
1.
INTRODUCTION
The use of nanofibers as carrier is promising for biomedical and agricultural
applications[1, 2]. In controlled and sustained release applications, the active chemicals
are dissolved in the polymer solution and incorporated into nanofibers by using a onestep method like electrospinning[3, 4]. Compared to the traditional bulk materials, these
nanostructured membranes largely facilitated the drug delivery processes in many
aspects of pharmacology[5]. The fibrous structure possesses the advantages of having a
three-dimensional open porous medium with high surface area, what improves
therapeutic efficacy and reduces toxicity[6, 7]. Due to the high surface area and the
porous structure of these nanofibers applications in drugs delivery systems such as the
liberation of anti-neoplastic[8], antibiotics[9], and pheromone agents have been
suggested[6]. Other appealing advantages reported to nanofibers obtained by
electrospinning are high encapsulation efficiency, high loading capacity, and costeffectiveness[8]. Solution blow spinning (SBS) is an alternative method to obtain
polymeric nanofibers[10, 11]. This technique produces fibers in the same size range as
electrospinning, although with a greater potential for commercial scale-up[10].
Compared to the electrospinning, SBS is faster and operates at higher injection rates.
Furthermore, SBS process does not use high voltage or electrically conductive collector
to generate nanofibrous membranes[12, 13]. Like electrospinning, SBS of active
agents in polymer solutions is expected to result in nanofibers with high surface area,
fundamental to enhance the water solubility and the dissolution rate of chemicals[14].
Moreover, these can be designed to provide rapid, immediate, or delayed dissolution
with sustained release behavior. The aforementioned features along with the ability to
spin several polymers without altering the basic setup make the SBS fibers an attractive
technology for application in delivery systems.
Biopolymers have been extensively used as material for drugs encapsulation due
to their biocompatibility[15]. Among these polymers, poly(lactic acid) is widely used
in medical applications such as drug delivery devices, absorbable sutures, and as
materials for medical implants and other related applications[16-18]. Poly(lactic acid)
has received great attention because its monomer, L-lactic acid, can be obtained by
fermentation from renewable resources such as starches and sugars[16].
The release of hydrophobic active agents from electrospun PLA fibers is
reported to follow a nearly zero-order kinetics due to the degradation mode of the
fibers[14]. Moreover Zeng et al.[19] registered a burst release for hydrophilic active
agents in PLA fibers via diffusion of the drug on or nearby the fiber’s surfaces.
Zein is considered a prolamine, the major storage protein of corn, which
comprises about 45–50% of its protein content[20]. Isolated, zein is not used directly for
human consumption due to its negative nitrogen balance and poor solubility in water.
As a biopolymer which provides many advantages in such terms as biocompatibility,
biodegradability, electrospinnability, and film forming ability, it is considered a
potential raw material for bioengineering applications[21].
One interesting agent to be tested in such delivery system is the progesterone
that consist in a progestin involved in their pregnancy[22]. In veterinary medicine,
progesterone is used as a potent drug for suppression of estrous and ovulation, making
possible the synchronization of the estrous and ovulation cycles in livestock
animals[23]. Currently, the estrous control of livestock animals is conducted by the
inserption of homogenous dispersions of progesterone embeds in silicone bands.
However, the disadvantages of these inserts, according to Rathbone et al.[23] lie in the
use of nondegradable polymers and the requirement for a disposal of these bands after
their use. Thus, the association of progesterone and poly(lactic acid) matrix can be an
excellent options for a bioabsorption material, such as for the manufacturing of
intravaginal drug delivery systems[16].
In this sense, the present study aims to investigate the morphology
characteristics of medroxyprogesterone encapsulated in poly(lactic acid) fibers obtained
by solution blow spinning, and compared with those on zein electrospun fiber
considering the effect of P4 addition on the morphology and structural fiber properties.
2.
MATERIALS AND METHODS
2.1
Materials
Poly(lactic acid) (PLA, Mn =75,000 g/mol) was obtained from Biomater Co.
(Brazil). Zein from maize and progesterone (P4, 99%) from Sigma-Aldrich (USA).
Chloroform, alcohol and acetone, used as solvent, were purchased from Synth (Brazil).
Dialysis membranes (cut-off 12,000 Da) were purchased from Sigma-Aldrich (USA).
2.1
Experimental
Preparation of Polymer Solutions
The solution formulations for SBS were prepared by weighting amounts of PLA,
zein and P4 according to proportions listed in tables 1and 2. Dissolution was carried out
in chloroform:acetone at 3:1 (v/v) for PLA and alcohol : water at 8:2 (v/v) for zein
under vigorous stirring for several hours at room temperature, until complete
dissolution.
Table 1 Poly(lactic acid) solution concentration of spun fibers.
PLA
Concentration (wt.%)
P4
Concentration (w/w %)
6
0
6
2
6
4
6
8
Table 2 Zein solution concentration of spun fibers.
Zein
Concentration (wt%)
20
P4
Concentration (w/w %)
20
2
20
4
20
8
0
Characterization of Solutions
For characterization of solutions the intrinsic viscosity of solution (η) was
determined for all polymer concentrations at 25 oC in a range of 10-100 s-1 shear rate,
using an Anton Paar Physica MCR rheometer. Concentric cylinders geometry (R = 24
and 28 mm diameters) were used. All measurements were performed in triplicate and
values expressed as the mean.
Fiber Spinning
Zein fibers prepared by electrospinning were spun using a voltage of 24 kV,
working distance of 12 cm, and a feed rate of 2 µL.min-1. Poly(lactic acid) fibers
obtained by solution blow spinning were prepared under an air pressure of 0.4 MPa,
working distance of 12 cm, and feed rate of 120 µL.min-1. Experimental operation
details can be found elsewhere[10, 11]. In both cases, the generated nanofibers were
collected on a rotating drum (200 rpm), wrapped with aluminum foil, generating
nonwoven fiber mats. The fiber mats were than collected by peeling off and stored in a
desiccator.
Characterization of Fibers
Fiber morphology was observed using a JEOL model JSM-6510/GS scanning
electron microscope (SEM). Samples were prepared by cutting samples of the mats with
a blade and fixed on aluminum stubs using double-sided adhesive tape and coated with
gold using a sputtering (Balzers, SCD 050). The diameter of the fiber was measured
with the aid of an image analysis software (Image J, National Institutes of Health,
USA). For each experiment, the average fiber diameter and distribution were
determined from approximately 100 measurements randomly taken from representative
fiber morphology.
Thermal analysis was conducted by differential scanning calorimetry (DSC) in a
Q100 TA Instruments. Samples were crimped in aluminum pans and heated at a rate of
10oC/min, from 0oC to 180oC in a nitrogen atmosphere.
3.
RESULTS AND DISCUSSION
3.1
Rheology
The viscosity data for each of the polymeric solutions in is presented in Fig. 1,
revealing differences in their properties as a function of hormone concentration.
Viscosity (mPa.s)
40
36
zein
zein+2% P4
zein+4% P4
zein+8% P4
32
28
0
20
40
60
80
100
-1
Shear rate (s )
(a)
(b)
Figure 1. Variation in viscosity as a function of the shear rate for PLA (a) and zein (b)
solutions.
Viscosity curves for polymer solutions of neat PLA in chloroform:acetone at 3:1
(v/v) and zein in alcohol:water at 8:2 (v/v), revealed a Newtonian behavior, as well as
for samples with 2 and 4% of progesterone added in PLA and 2,4 and 8% of
progesterone added in zein. In general, the viscosity of the PLA and zein solutions
increased as the concentrations of progesterone increases. Moreover, a pseudoplastic
behavior was observed to PLA+ 8% P4 samples. The effect of the presence of
progesterone in increasing PLA and zein solutions viscosity can be explained in terms
of the interaction forces taking place between the progesterone and the biopolymer.
Depending on the type and intensity of these interactions, rheological properties of
polymer solutions can changed and thus affect the releasing behavior when in the solid
state. The increase in the viscosity of PLA and zein solutions by the addition of
progesterone indicates interactions between the polymer and the progesterone.
3.2
Poly(lactic acid) fiber mophology
Figure 2 shows SEM images of solution blow spinning nanofibers obtained from
PLA plus progesterone additions of 2; 4 and 8 %wt as collected on the rotating drum.
1 μm
(a)
(b)
(c)
(d)
Figure 2. Poly(lactic acid) fiber morphology: (a) neat, (b) 2%P4, (c) 4% P4 and (d)
8%P4
Solution blow spun fibrous membranes with uniform morphology and a narrow
size distribution were produced. The hormone-free and the hormone loaded PLA fibers
have both a smooth surface and no progesterone crystals were observed on fiber surface.
This suggested that progesterone was dispersed homogeneously in the solution and into
the spun fibers. The average diameters ranged from 289 nm for PLA fibers to about 440
nm for fibers with progesterone (Table 3). The increase in the average fiber diameter
due to active agents addition can be interpreted as a success in the incorporation of P4 in
the PLA matrix.
Table 3 Solution Blow Spinning fiber diameter
Fiber
Progesterone added
(% wt)
PLA
0
PLA
2
PLA
4
Resulting fiber diameter
(nm)
289 ± 92
283 ± 80
441 ± 180
PLA
3.3
8
398 ± 220
Zein fiber morphology
Figure 3 shows SEM images of electrospun nanofibers obtained from zein plus
progesterone additions of 2; 4 and 8 %wt.
(a)
(b)
(c)
(d)
Figure 3. Zein fiber morphology: (a) neat, (b) 2%P4, (c) 4% P4 and (d) 8%P4
Neat zein fibrous membranes with uniform morphology and a narrow size
distribution were produced. As PLA case, the hormone-free and the hormone loaded
zein fibers have both a smooth surface and no progesterone crystals were observed on
fiber surface. This suggested that progesterone was dispersed homogeneously in the
solution and into the electrospun fibers. Moreover, beaded fibers were observed to
zein+ hormone samples. The effect of the presence of progesterone in increasing bead
concentration in zein fibers can be explained by polymer/drug/solvent interactions.
Progesterone is soluble in alcohol phase but not in water phase. This fact leads changes
in rheological properties of these solutions that cannot be observed in PLA+
progesterone solutions.
The average diameters ranged from 146 nm for zein fibers to about 79 nm for
fibers with progesterone (Table 4). The decrease in the average fiber diameter due to
active agents addition can be interpreted as increase in the bead concentration.
Table 4 Electrospun fiber diameter
Fiber Progesterone added
Resulting fiber diameter
(% wt)
(nm)
zein
0
146 ± 33
zein
2
143 ± 46
zein
4
95 ± 25
Bead Concentration
(beads/µm2)
0
0.04
0.2
zein
3.4
8
79 ± 29
1
Thermal Properties
DSC was used to determine melting temperature (Tm), glass transition
temperature (Tg), and the heat of fusion (ΔHf) for neat PLA and zein nanofibers and
biopolymer/progesterone nanofibers as shown in tables 5 and 6.
Table 5. Characteristic temperatures and heat of fusion (ΔHf) for solution blow spinning
nanofibers
PLA Fibers
Thermal
Properties
Neat
2% P4
4% P4
8%P4
Tg (oC)
59
57
56
56
Tm (oC)
150
145
147
147
ΔHf (J/g)
23
20
21
21
For PLA fibers both melting (Tm) and glass transition temperature (Tg) decreases
slightly as the progesterone content increases. In general, reductions observed in the
melting point in a polymeric blend are normally associated to alteration on
morphological effects (e.g., a decrease in lamellar thickness) and to a weak polymerpolymer interactions. In other words, the progesterone seems to have a plasticizer
behavior within the PLA polymer matrix. Similarly, the change in glass transition
temperature is generally explained as a consequence of polymer/plasticizer interactions
such as due to dipole forces. In particular, a decrease in Tg is also associated to
plasticizer effect of the additive.
Table 6. Characteristic temperatures and heat of fusion (ΔHf) for electropsun nanofibers
Zein Fibers
Thermal Properties
Neat
2% P4
4% P4
8% P4
Tg (oC)
130
137
137
138
Tm (oC)
83
79
78
74
ΔHf (J/g)
114
110
111
36
Glass Transition temperature for zein fibers was increased as the progesterone contente.
Probably, this change can associated to alteration on protein structure of zein.
Interactions between zein and progesterone lead to a decrease of segmental mobility of
macromolecules. Water melting temperature and heat of fusion are sightly decreased by
progesterone.
3.5
Hormone Release
Figure 4 illustrates the release behaviors of progesterone from the
nanostructured PLA membranes. It is observed that the difference between the PLA/P4
systems releasing is basically related to the total amount of progesterone released (i.e., a
determined amount of PLA+8%P4 will release more progesterone in the media than the
same quantity of PLA+4%P4, which, consequently, will release more progesterone in
the media than the PLA+2%P4 fiber). However, the release comportment of each
system is basically the same, obeying a first order kinetic of liberation with very similar
K constant values. Moreover, Fig. 4 showed that the half-life for all samples is between
4 and 5 hours.
Figure 4. In Vitro release profile of progesterone from PLA membranes.
4.
CONCLUSIONS
The results of the current study confirm some miscibility of progesterone and
hydrophobic biopolymers according to rheology, and thermal used methods.
Progesterone was found to have the effect as plasticizer when added to PLA nanofibers.
The experimental data display a decrease in the Tg values, which is generally attributed
to polymer/active agent interactions. Thus, nanofiber technology as electrospinning and
solution blow spinning can be appropriated used to encapsulate active agents in
biodegradable and biocompatible polymers, providing a release with first-order kinetics
rates. This study clearly indicated that PLA and zein nanofibers can be potentially used
in controlled delivering system of progesterone, being a powerfull to in the control of
the estrus cycle in livestock animals.
ACKNOWLEDGEMENTS
The authors are grateful for the financial support from FINEP/MCT, CAPES,
CNPq, Embrapa/LNNA/Rede AgroNano, and FAPESP (Process No 2010/19860-1)
funding agencies.
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