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Corresponding author. Tel.: +55 15 3238 3414; fax: +55 15 3228 2842.
E-mail address: leonardo@sorocaba.unesp.br (L.F. Fraceto).
1. Introduction
The herbicides used in agriculture have become a multi-billion
dollar global industry. Brazil is the largest single country user
of these chemicals, with over a million tons applied to cultiva-
tions every year [1]. Triazines are one of the most commonly used
0304-3894/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2012.06.019
2 R. Grillo et al. / Journal of Hazardous Materials 231232 (2012) 19
classes of herbicides, and are widely employed to control weeds
in maize, sorghum, and sugar cane plantations. The class includes
ametryn (AMT), atrazine (ATZ), and simazine (SIM), whose prop-
erties include high runoff potential, prolonged persistence in soils,
slowhydrolysis, lowvapor pressure, lowwater solubility, andmod-
erate adsorption onto soil organic matter and clays [2].
Despite their widespread adoption in agriculture, these herbi-
cides can be dangerous to humans, depending on their toxicity, the
degree of contamination, and the duration of exposure. The inap-
propriate use of herbicides cancontaminate air, soil, andwater, and
lead to the extinction of sensitive species [3].
Controlled release systems are increasingly used to miti-
gate problems of toxicity, minimize environmental impacts, and
increase efciency. Materials used in agriculture for this purpose
include liposomes [4], cyclodextrins [57], clays [810], silica [11],
lignin [12,13], polymeric microparticles [1418] and nanoparticles
[19,20]. Biodegradable micro and nanoparticles are especially suit-
able as carriers for pesticides, due to their low toxicity and good
biocompatibility.
Poly(-caprolactone) (PCL), a biodegradable polymer widely
used in controlled release systems, is an aliphatic polyester able
to formmicro- and nanoparticles. It is insoluble in water (although
water can permeate the polymer chains), slowto degrade in aque-
ous media, and harmless in the environment [21]. Sinha et al. [22]
described the use of various procedures by which the polymer can
incorporate pharmaceuticals. Two types of nanoparticles can be
produced, depending on the materials and the procedure adopted.
The nanoparticles denition can be considered very broad because
not only depends on the size of below 100nm, but also the appli-
cation of this system in the medical or agricultural. An important
factor toconsider is whether the particles are governedbyquantum
effects instead of Newtonian physics. The ability to differentiate
between quantum effects and Newtonian physics is based on a
comparison of the size of the particles to the wavelength of their
motion[23]. Nanoparticles canbe foundintwoways: nanocapsules
(NC) are composed of a polymeric shell and an oily nucleus, while
nanospheres (NS) consist of a dense polymeric matrix without any
oil [16,2426].
Considering the widespread global use of herbicides, the appli-
cation of controlled release systems is of interest from both
ecological and economic perspectives. The objective of this work
was to prepare and characterize such a release systemfor use with
the triazine herbicides ametryn, atrazine, and simazine. The her-
bicides were encapsulated in poly(-caprolactone) nanocapsules,
and the genotoxicities of the formulations were evaluated. The sys-
tem was designed in order to help minimize the impacts of the
herbicides on human health and the environment, when used in
plantations.
2. Experimental
2.1. Materials
Ametryn, atrazine, simazine, poly(-caprolactone), Tween 80
and Span 60 were purchased from SigmaAldrich. The solvents
used in the chromatographic analyses were HPLC-grade acetoni-
trile (fromJ.T. Baker) and Milli-Qwater. The solutions were ltered
using 0.22m nylon membranes (Millipore, Bedford, USA). All
reagents were obtained fromlocal suppliers at analytical grade or
better, if not otherwise stated in the text.
2.2. Preparation of the polymeric nanocapsules containing the
triazine herbicides
The PCL nanocapsules were prepared according to the interfa-
cial deposition of pre-formed polymer method, rst described by
Fessi et al. [27], which involves the mixing of an organic phase into
an aqueous phase. The organic phase was composed of 100mg of
polymer (PCL), 30mL of organic solvent (acetone), 200mg of oil
(triglycerides of capric and caprylic acids, in the form of Miglyol
810), 40mg of sorbitan monostearate surfactant (Span 60), and
10mg of herbicide (ametryn, atrazine, or simazine). The aqueous
phase was composed of 30mL of a solution containing 60mg of
polysorbate 80 surfactant (Tween 80). After dissolving the compo-
nents of bothphases, the organic phase was slowlyinsertedintothe
aqueous phase, with magnetic stirring. The resulting suspension
was maintained under agitation for 10min, after which the organic
solvent was evaporated under reduced pressure using a rotary
evaporator [27,28]. The nanoparticle suspension was evaporated
to a nal volume of 10mL, resulting in an herbicide concentration
of 1mg/mL. The formulations were stored in amber asks at room
temperature (25
C).
2.3. Association efciency measurements
The total (100%) amounts of the herbicides present in the NC
suspensions were determined by high-performance liquid chro-
matography (HPLC), after dilution in acetonitrile and ltration
through a 0.22mMillipore membrane.
The amounts of the herbicides associated with the NC were
measured using the ultraltration/centrifugation method, which
consisted of centrifuging the NC suspension in ultraltration
vessels composed of 30kDa regenerated cellulose (Microcon, Mil-
lipore), and then analyzing the ultraltrates by HPLC. Only the
free herbicides passed through the 30kDa membrane, so that the
amounts associated with the nanoparticles could be calculated
by difference. Quantication employed previously validated cali-
bration curves describing the relationships between the herbicide
concentrations and the HPLC detector responses. Several earlier
studies have used the same procedure to measure the association
efciencies of compounds innanoparticles [12,21,2932]. TheHPLC
analyses were performed using a Varian
ProStar instrument t-
ted with a PS 210 pump, a PS 325 UVvis detector, a Metatherm
0.22mmembranes.
2.4. Release kinetics assays
The kinetics experiments designed to examine the release
proles of the herbicides employeda systemconsistingof twocom-
partments (donor and receptor), maintained under gentle agitation
[31]. A cellulose membrane (Spectrapore, with 1000Da molecu-
lar exclusion pore size) separated the sample (2mL) in the donor
compartment from the receptor compartment containing 100mL
of water. The pore size of the membrane ensured that only the free
herbicides passed through to the receptor compartment, while the
herbicides associated with the NC were retained in the donor com-
partment until theshift inequilibriumresultedintheir releasefrom
the interior of the NC. The experiments were performedunder dilu-
tion sink conditions, and samples were collected fromthe receptor
compartment after different time intervals, for analysis using HPLC.
All measurements were performed in triplicate.
2.4.1. Release efciency (RE)
The concept of release efciency can be a useful means of eval-
uating release kinetics. It is dened as the area under the release
curve during a specic time interval [33]. Here, RE was calculated
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R. Grillo et al. / Journal of Hazardous Materials 231232 (2012) 19 3
fromthe areas under the herbicide release prole curves, for differ-
ent time intervals (t), using the trapezoidal method of integration.
2.4.2. Mathematical modeling of the release experiments
The proles of the release of biologically active compounds from
nanoparticles can provide important information concerning the
mechanisms involved. Advances in information technology have
led to the creation of new mathematical models that enable pre-
diction of these mechanisms [34,35].
The semi-empirical KorsmeyerPeppas model (Eq. (1)) was
applied to the herbicide release curves in order to identify the type
of mechanisminvolved [31].
M
t
M
= Kt
n
+b (1)
Here, M
t
amount of herbicide releases at time t, M
total amount of
herbicide releases at time t, K is a constant that considers structural
and geometrical aspects of the system, b is the initial amount of
herbicide in solution and the value of the exponent (n) is used to
characterize the release mechanism. The model analysis employed
the values obtained from the linear regressions for the different
treatments.
2.5. Physico-chemical stability of the nanocapsules
Thestabilities of thesuspensions of NCcontainingtheherbicides
were evaluated frommeasurements of particle diameter, polydis-
persity, zeta potential, and pH, over a period of 270 days storage of
the suspensions in amber asks, at roomtemperature (25
C).
2.5.1. Size and polydispersity measurements
The dynamic light-scattering technique was used to measure
the average size (hydrodynamic diameter) of the particles, and
their polydispersity. The NC suspensions were diluted (1:100, v/v),
and measured (at 25
C) using a Malvern
. The results
were expressed as the means of ten readings.
2.5.2. Zeta potential measurements
The zeta potential values (in units of mV) were also obtained
using the Malvern
C,
ina solutionof 1MHCl. Colorationwas performedusing Schiff solu-
tion, in the absence of light, for 2h. The slides were prepared using
the meristematic region of the root tip, with application of a drop
of 2%acetic carmine, and covering with a cover slip which was used
to squash and spread the cells. Around 500 cells were observed on
each slide, using an optical microscope.
During the treatments, the samples were exposed to different
concentrations of ATZ, AMT, and SIM (1, 10, and 100mg/L), either
free or encapsulated in the PCL nanocapsules. The controls used
were deionized water, PCL nanocapsules without herbicide, and
dimethylsulfoxide (used to solubilize the unencapsulated herbi-
cides).
The data analysis involved calculations of the mitotic index,
which considers the proportion of cells in division, and the
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4 R. Grillo et al. / Journal of Hazardous Materials 231232 (2012) 19
Fig. 1. Results of in vitro release kinetics experiments for ametryn (AMT), atrazine
(ATZ), and simazine (SIM) contained in PCL nanocapsules, at 25
C (n=3).
damage index, which considers changes that occur during the
prophase, metaphase, anaphase, and telophase stages of growth.
3. Results and discussion
3.1. Determination of encapsulation efciency
Particles in the nanometric size range have a high surface area
to volume ratio, which allows molecules to be readily encapsu-
lated in their interiors. Nonetheless, consideration is needed of
the chemical nature and polarity of the compound to be encapsu-
lated, since hydrophilic compounds have lowassociation afnities
(10%), while hydrophobic substances have high afnities (>70%)
[25]. The herbicides used here have low water solubility, and the
association values obtained were 84.12.5% (AMT), 86.70.7%
(ATZ), and 97.00.5% (SIM). All the compounds showed good
association efciencies, demonstrating their afnity for the nanos-
tructured system.
3.2. Release kinetics assays
In matrix-based systems, various parameters can inuence the
release of herbicides, including particle size, molecular weight,
and polymer ratio. Release kinetics experiments are a fundamental
means of obtaining information concerning the herbicidecarrier
interaction, as well as the mechanismof the release process [21,30].
The HPLC results were plotted as the percentage of herbicide
released fromthe nanocapsules as a function of time (Fig. 1). Since
only the herbicide molecules were able to traverse the pores of
the membrane, it was therefore possible to observe the effect of
the degree of association on the rate of release fromthe polymeric
nanocapsule matrix.
Over the 5days of the study, the proles of the release of ATZand
AMT fromthePCL nanocapsules wereslightlydifferent (Fig. 1), with
maximumrelease rates of 72 and 61%, respectively. The efciencies
of encapsulationof these twoherbicides were verysimilar, at 85%,
whichindicates that the differences inthe release proles canprob-
ably be explained by the different degrees of interaction between
the herbicide molecules and the oily and/or polymeric phases [25].
Compared to SIM, the chemical structures of ATZ and AMT contain
a greater quantity of CH
3
groups, which can cause steric hindrance
andinhibit the formationof hydrogenbonds betweenthe herbicide
molecules and the polymeric chains. This diminishes the interac-
tion, and hence accelerates the release of the bioactive compound.
The release of simazine fromthe nanocapsules was muchslower
than that of the other two herbicides, with only 25% released after
Fig. 2. Results obtained using the Korsmeyer-Peppas mathematical model applied
to ametryn (AMT), atrazine (ATZ), and simazine (SIM) contained in PCL nanocap-
sules. M represents the absolute amount of herbicide released in time t, and M
100%
is the total amount of herbicide released after innite time.
5 days. This difference could be related to the high hydrophobicity
of simazine, as well as the stronger interaction of the compound
with the PCL polymeric chains due to the smaller number of CH
3
groups, compared to the other herbicides.
Integration of the release proles gave release efciencies (RE)
of 56.5, 47.2, and 18.7% for the ATZ, AMT, and SIM formulations,
respectively. This procedure provided a more reliable compari-
son of the relative rates of release of the herbicides from the PCL
nanocapsules, with values decreasing in the order ATZ>AMT >SIM.
3.2.1. Mathematical models
The release of herbicides from polymeric nanoparticles is gov-
erned by various mechanisms, including desorption from the
surface, diffusionthroughthe pores or wall of the polymeric matrix,
and disintegration, dissolution or erosion of the polymeric struc-
ture [25]. Identication of the mechanisms involved can be greatly
assisted by the use of mathematical models [31], and the model
described by Korsmeyer and Peppas [38] has been widely used
to identify the type of mechanism whereby pharmaceuticals are
released from nanostructured systems. This model is appropriate
in situations where the release mechanismis not well understood,
or where more than one mechanismis involved, such as a combi-
nation of active diffusion (Fickian transport) and Type II transport
(non-Fickian, controlled by relaxation of the polymer chains) [38].
Korsmeyer andPeppas [38] proposedvalues of the release expo-
nent (n) that characterized three different mechanisms (Fickian,
anomalous, or Type II transport). According to Korsmeyer and
Peppas [38], n<0.43 indicates that release is governed purely by
classical Fickian diffusion, while n>0.85 reects Type II transport,
involving polymer swelling and relaxation of the polymeric matrix.
Intermediate values of n (0.43<n<0.85) indicate anomalous trans-
port kinetics, with a combination of the two diffusion mechanisms
and Type II transport.
The release prole data were treated using the
KorsmeyerPeppas model (Fig. 2) in order to obtain the val-
ues of the release constant (K) and the exponent (n) (Table 1).
Table 1
Values of the release constant (K), exponent (n), correlation coefcient (r), and
release efciency (RE) for the herbicides contained in PCL nanocapsules.
Sample K n r RE
PCL-AMT 3.210
4
min
1
0.997 0.998 47.2%
PCL-ATZ 6.810
4
min
1
0.924 0.924 56.5%
PCL-SIM 2.410
4
min
1
0.871 0.998 18.7%
R. Grillo et al. / Journal of Hazardous Materials 231232 (2012) 19 5
For all three herbicides, values of n were in the range 0.870.99,
indicating that the release mechanism involved Type II transport
(non-Fickian, controlled by relaxation of the polymer chains). The
values of K were 6.810
4
min
1
(ATZ), 3.210
4
min
1
(AMT),
and 2.410
4
min
1
(SIM).
3.3. Evaluation of the stabilities of the nanocapsule formulations
The stabilities of the NC suspensions were determined using
measurements of their physico-chemical properties (diameter,
polydispersity, and zeta potential), as a function of time (0, 15, 30,
60, 90, and 270 days). The samples were stored in amber asks at
roomtemperature (25
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8 R. Grillo et al. / Journal of Hazardous Materials 231232 (2012) 19
The present results revealed signicant differences between
the treatments and the controls (p<0.05). Notably, there were
also signicant differences between the effects of the herbicides
used either alone or in association with the nanocapsules, with
encapsulation resulting in lower toxicity (p<0.05). The effect of
encapsulation was greatest in the case of ametryn (Table 2), for
which there was a reduction of around 50% in the damage index
value when the herbicide was encapsulated, indicating that there
was a decrease of 50% in cell DNA damage.
In earlier work by our research group [15,45], Allium cepa and
Lactuca sativa were used to assess the genotoxicity of ametryn and
atrazine, used either alone or in microencapsulated form, and a
similar reduction in toxicity was observed following encapsulation
of the compounds.
The Alliumcepa analysis enables calculationof the mitotic index,
which provides an indication of compound cytotoxicity. A decline
in the mitotic index below22%, relative to the control, can be lethal
to the organism[46]. Falls greater than 20% were observed for the
treatments using the herbicides alone, compared to both the con-
trol and the treatments using the encapsulated compounds. This
shows that encapsulation of the herbicides in the PCL nanocap-
sules maintained the mitotic indices at levels similar to that of the
control (Table 3).
The Alliumcepa analyses were also used to determine the chro-
mosome aberration indices for the different treatments. Greater
index values were obtained for the treatments using the herbi-
cides alone, compared to the encapsulated compounds, while in
the latter case the index values were similar to that of the negative
control (Fig. 6). These results are in agreement with the ndings
of Grillo et al. [15], where the Lactuca sativa test revealed reduced
genotoxicity of atrazine after encapsulation in microspheres.
4. Conclusions
The results of the present work showthat poly(-caprolactone)
nanocapsules can provide a useful modied release system for
the triazine herbicides ametryn, atrazine, and simazine. Encapsu-
lation efciencies exceeding 84% were achieved using suspensions
of the nanocapsules. The kinetic proles obtained demonstrated
that release of the herbicides from the nanocapsules was slower,
compared to the free herbicides, and that the mechanismof release
was associated with relaxation of the polymeric chains. The sizes
of the nanocapsules containing the herbicides were in the range
232290nm, the polydispersion values were below 0.2, and zeta
potentials were around 30mV. The formulations remained sta-
ble for at least 270 days. Microscopy analyses showed that the
nanocapsules were spherical, with a uniformsize distribution and
no apparent aggregate formation. The results of infrared spec-
troscopy indicated that the herbicide molecules interacted with
the nanocapsule polymers. Genotoxicity tests using different cell
types (human lymphocytes and Allium cepa cells) indicated that
the encapsulated formulations were less toxic than the herbicides
used in their free forms. The nanocapsule carrier system offers an
attractive alternative method for the sustained release of triazine
herbicides, which could help to mitigate environmental impacts, as
well as reduce the quantities of chemicals requiredfor weedcontrol
in plantations.
Acknowledgements
The authors thank FAPESP, CNPq, and FUNDUNESP for nancial
support.
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