Ticks and Tick-borne Diseases 13 (2022) 101849

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

journal homepage: www.elsevier.com/locate/ttbdis

Chitosan-coated poly (Ɛ-caprolactone) nanoparticles as acaricide carriers

Elias Berni a, Raquel de Melo Barbosa b, c, *, Nelson Durán a, d, e
a
Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, CEP 13083-970, CP 6152, Campinas, SP, Brazil
b
Biochemistry Department, Institute of Biology, Universidade Estadual de Campinas, Campinas, SP, Brazil
c
Department of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil
d
NanoBioss (MCTI), Institute of Chemistry, Universidade Estadual de Campinas, Campinas, SP, Brazil
e
Brazilian Nanotechnology National Laboratory (LNNano-CNPEM), Campinas, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Among many species of ticks that affect beef and dairy cattle, Rhipicephalus (Boophilus) microplus is the most
Polymeric nanoparticles common. It is responsible for heavy losses in milk and meat production. In this work we introduce nanostructures
Ticks such as chitosan-poly-Ɛ-caprolactone (CS_PCL) nanoparticles to encapsulate amitraz (CS_PCLnp_Amitraz) and
Amitraz
fluazuron (CS_PCLnp_Fluazuron) to treat tick infestations more effectively. The CS_PCLnp_Amitraz system has a
Fluazuron
Poly-ε-caprolactone
final amitraz concentration of 1.0 mg/mL with a particle size of 275 ± 30 nm, surface charge of +43 ± 7 mV and
entrapment efficiency of 77 ± 1%. The CS_PCLnp_Fluazuron system has a drug concentration of 0.5 mg/mL with
a particle size of 295 ± 35 nm, surface charge of +45 ± 10 mV and entrapment efficiency of 89 ± 1%. Both
systems reduced cytotoxicity on Balb/c 3T3 culture cells and were also active against R. microplus. Both mole­
cules - amitraz and fluazuron – formed molecularly dispersed active compounds inside the core of the PCL
polymer matrix. The PCL surface was composed of a chitosan layer, which influenced the stability of the steric
nanoparticles at pH greater than 7. Both systems were stable at a saline concentration of 1.25 mol/L and at
temperatures below 50 ◦ C. Experiments conducted in vivo with CS_PCLnp_Amitraz, at doses of active ingredient
equivalent to those of commercial products, showed decreased tick infestation for 21 days, as well as higher
acaricide effect than observed for commercial products, which recommend a reapplication in 14 days. The
acaricide effect was even stronger when CS_PCLnp_Amitraz (same dose as for commercial products) and
CS_PCLnp_Fluazuron (half of the amount for commercial products) were administered together.

1. Introduction into consideration the economic losses caused by R. microplus when

Rhipicephalus (Boophilus) microplus, also known as the Asian blue
tick, is the most common tick species affecting beef and dairy cattle. It
accounts for significant losses in milk and meat production, which can
be classified as i) direct losses due to blood spoliation and its conse­
quences such as anemia, itching, irritation, predisposition to larval
infestation and decreased animal weight; and ii) indirect losses due to
protozoan parasites, such as Babesia spp., transmitted by this tick species
and causing diseases in cattle (Dantas-Torres et al., 2012; Jongejan and
Uilenberg, 2004; Madder et al., 2011; Wu et al., 2013). Rhipicephalus
microplus is widely distributed in the tropics and subtropics (Ren et al.,
2012). Economic losses caused by tick infestations are significant
(Beltrão et al., 2013; Grisi et al., 2002; Walker, 2011). Current data refer
to economic loss of US$42.58/animal in Brangus cattle and in its crosses
(Calvano et al., 2019; Matzembacker et al., 2019). It is essential taking
analyzing cattle production systems (Andreotti et al., 2016). Estimates
have suggested that the total economic loss accountable to R. microplus
infestations in Brazilian cattle herds is approximately US$ 3.2 billion per
year (Grisi et al., 2014).
Despite the significant economic losses caused by R. microplus in­
festations, the problem has not been given the attention it deserves.
Arsenicals were the first compounds used against ticks, in the late 1940s,
later followed by organophosphates and organochlorines (which act on
ticks’ neuromuscular system). Other compound families then emerged,
namely: formamidine, in the 1970s; pyrethroids, in the 1980s; and
benzoyl-phenyl urea, in the 1990s (Plimmer et al., 2003). Unfortunately,
improper acaricide management has generated environmental pollu­
tion, livestock food contamination, and increased acaricide doses due to
the tick becoming resistant during treatment. Besides, there is a high loss
of revenue on account of production decrease and high animal treatment

* Corresponding author.
E-mail address: raquel.barbosa@dca.ufrn.br (R.M. Barbosa).

https://doi.org/10.1016/j.ttbdis.2021.101849
Received 8 October 2020; Received in revised form 27 August 2021; Accepted 22 September 2021
Available online 10 October 2021
1877-959X/© 2021 Elsevier GmbH. All rights reserved.
E. Berni et al.
cost (Ángel-sahagún et al., 2010; Pourseyed et al., 2010; Rosa et al.,
2012). Amitraz and fluazuron, which were developed to treat ticks such
as R. microplus, are some of the most used acaricides (Dos Santos et al.,
2009). Amitraz acts by inhibiting the enzyme monoamine oxidase ac­
tivity, causing a hyperexcitation that promotes the parasite to paralyze
and detach it from the host (Filippo et al., 2006; Prullage et al., 2011).
Conversely, fluazuron inhibits chitin synthesis and thus interrupts the
tick life cycle at various stages (Akre, 2017).
Control of R. microplus remains a significant challenge in several beef
producing countries. The development of more effective acaricides is
one of the best alternatives to help mitigate issues associated with
acaricide resistance in ticks and high toxicity levels of these compounds.
In the last decade, there has been a remarkable increase in the number of
innovative nanobiotechnological products with applications in medi­
cine, biomedicine, food engineering, pharmacology, parasitology and
entomology (Kaushik, 2019; Patra et al., 2018; Periyannan et al., 2017;
Thiruvengadam et al., 2017). Veterinary medicine products incorpo­
rating nanomaterials have been used for disease diagnosis, treatments
based on controlled drug release, specific targeting, toxicant reduction,
as well as for agricultural supply monitoring and traceability purposes
(Ferreira et al., 2020; Kumar et al., 2019).
Nowadays, the improvement of existing products is the most widely
used alternative to them (Behrenz and Schutte, 1989; Bouvier and Kolly,
2005). This procedure can be followed either by using two or more
molecules with different actions (Hunter et al., 2011) or by using
nanoparticles in different targeted delivery ways (Marcato and Durán,
2008). The use of nanoparticles in drug delivery processes is well known
in the medical field; Doxil® and Abraxane® stand out among the several
products available in the market. However, there is significant lack of
nanotechnology-based drugs for veterinary purposes in comparison to
products developed for human pharmacotherapy. This presents an op­
portunity for innovation in profitable and necessary pharmaceutical
products focused on animal health (Ferreira et al., 2020; Gonçalves
et al., 2020).
In light of the foregoing, the aim of the current study was to combine
chitosan and poly (Ɛ-caprolactone) as an alternative for encapsulating
two known potent actives, amitraz (Jonsson et al., 2010) and fluazuron
(Kryger et al., 2007), to use this system as a treatment against
R. microplus (Duran et al., 2014). Chitosan was used on the nano­
particles’ surface specifically to provide mucoadhesive properties
(Mazzarino et al., 2012).
2. Materials and methods
2.1. Materials
Poly (epsilon-caprolactone) (PCL) (Mw=65,000 g/mol) was pur­
chased from Sigma Aldrich, Burlington, MA, USA. Chitosan (CS)
(Mw=65,000 g/mol and with a deacetylation degree of 81%) was pur­
chased from Polymar, Fortaleza, CE Brazil. Acetone and acetic acid were
purchased from Synth Ltda, Diadema, SP, Brazil. Amitraz and fluazuron
were purchased from Santa Cruz Biotechnology, Mississauga, ON,
Canada. Polyoxyethylene (20) sorbitan monolaurate (Tween 80) was
kindly provided by Oxiteno, Suzano, SP, Brazil. All reagents were used
without modifications.
2.2. Polymeric nanoparticle preparation
The solvent displacement method, also called nanoprecipitation, was
used to synthesize chitosan-coated polymer nanoparticles (Fessi et al.,
1989). Briefly, 125 mg of PCL were dissolved in 25 mL of acetone at
moderate temperature (~35 ◦ C) in order to synthesize unloaded nano­
particles. The organic phase was poured into 75 mL of the aqueous phase
added with 0.5% of acetic acid (v/v), 0.0425% of chitosan (w/v) and 1
mg/mL of Tween 80. Nanoparticles were formed right after organic
phase was poured into aqueous phase, after that acetone was eliminated
2
Ticks and Tick-borne Diseases 13 (2022) 101849
by evaporation under reduced pressure. In order to produce
drug-bearing nanoparticles, 75 mg of amitraz or 37.5 mg of fluazuron
were dissolved in acetone with PCL, by following the same synthesis
procedure applied to unloaded nanoparticles. Unloaded nanoparticles
were called Chitosan-PCLnp (CS_PCLnp), whereas nanoparticles loaded
with amitraz and fluazuron were called Chitosan-PCLnp-Amitraz
(CS_PCLnp-Amitraz) and Chitosan-PCLnp-Fluazuron (CS_PCLnp-Flua­
zuron), respectively.
2.3. Morphological featuring
The mean diameter, polydispersity index (PDI) and zeta potential of
the nanoparticles produced were investigated based on the dynamic
light scattering (DLS) technique in ZetaSizer Nano instrument, ZEN3600
(Malvern Instruments, Malvern, UK). Particle size distribution was
measured through nanoparticle tracking analysis (NTA) technique in
NanoSight LM10 equipment (NanoSight, Malvern, UK) (Filipe et al.,
2010). Ten-second-long videos were recorded with video camera, whose
parameters were set to get the best image. All samples were diluted in 1
mmol/L of KCl solution (1:19, v/v). Measurements were taken at 25 ◦ C.
The morphology of the nanoparticles was evaluated through trans­
mission electron microscopy (TEM), using a Libra 120 instrument (Zeiss,
Oberkochen, Germany). A drop of sample diluted in MilliQ® water (at
1:19 sample: water ratio) was placed on copper grid coated with formvar
film.
2.4. Nanoparticles’ stability
Shelf life is vital information to help determine the minimum
viability of any product (Brasil and MAPA, 2012). Besides, the current
study was designed to obtain high nanoparticle concentrations. The
acaricide products must be properly diluted and remain stable in any
water pH and/or salinity range just before being applied to tick-infested
animals in the field.
Thus, CS_PCLnp_Amitraz and CS_PCLnp_Fluazuron stability was
evaluated by monitoring the mean diameter, size, and zeta potential of
nanoparticles, under varying pH value, salt concentration, temperature,
and time after production. Changes in size and zeta potential of nano­
particles were monitored through the DLS technique; pH was modified
by external module, MPT – 2 (Malvern Instrument, Malvern, UK). Acidic
(0.25 mol/L of HCl) and basic (0.5 molL− 1 and 0.25 mol/L of KOH)
solutions were used to correct the pH.
2.5. Differential scanning calorimetry
The physical state of and interaction between compounds in nano­
particles were investigated based on differential scanning calorimetry
DSC-Q10 (TA Instruments, New Castle, DE, USA). First, samples were
lyophilized, placed in aluminum pans, and sealed. All samples were
heated from 20 ◦ C up to 200 ◦ C, at rate of 10 ◦ C/min and isothermally
held in nitrogen atmosphere (50 mL/min) for 1 min.
2.6. Loading efficiency and acaricide release in vitro
Entrapment efficiency and total amount of encapsulated amitraz or
fluazuron were analyzed in a liquid chromatographer coupled to a mass
spectrometer, LC-MS/MS Waters UPLC Acquity–TQD Quattro MicroAPI
(Waters, Milford, MA, USA). Amitraz (10 µg/mL) and fluazuron (20 µg/
mL) stock solutions were prepared to generate the calibration curves.
Both curves presented adjusted R2 values higher than 0.99; the amitraz
curve showed LD=0.29 µg/mL and LQ=0.31 µg/mL, whereas the flua­
zuron curve showed LD=1.64 µg/mL and LQ=1.68 µg/mL (LD is the
detection limit and LQ is the quantification limit). Moreover, 500 μL of
loaded nanoparticle suspension was centrifuged (14,500 rpm for 15 min
at room temperature) and the supernatant was removed for amitraz or
fluazuron amount determination. Pellets were dried, diluted in 500 μL of
E. Berni et al. Ticks and Tick-borne Diseases 13 (2022) 101849

Fig. 1. Transmission electron microscope images of CS_PCLnp_Amitraz (A) and CS_PCLnp_Fluazuron (B). The size distribution diameter of CS_PCLnp_Amitraz (C) and
CS_PCLnp_Fluazuron (E) were compared between two different techniques, dynamic light scattering (ZetaSizer) and nanoparticle tracking analysis (NanoSight), a
frame of data obtained with NTA analysis for CS_PCLnp_Amitraz (D) and CS_PCLnp_Fluazuron (F) is also presented.

acetonitrile and left to rest for 5 h. These solutions were placed in
Microcon® filter (10 kDa) and centrifuged as previously described; the
supernatant was measured through mass spectrometry. Tween 80 (10
mg/mL) was used to find the sink condition.
2.7. Cytotoxicity assays
Balb/c3T3 cells were provided by NIH (National Institute of Health,
Baltimore, MD, USA). Cells were grown in DMEM (Dulbecco’s modified
Eagle’s medium) supplemented with 10% fetal bovine serum, 1%
penicillin streptomycin solution (100 UI/mL of penicillin and 100 µg/
mL of streptomycin sulfate in 0.9% NaCl) in humidified atmosphere (5%
CO2 air) at 35 ◦ C. Cells were cultured in 75 cm2 cell culture flasks at 2 ×
104 cells/mL for Balb/c 3T3, at 37 ◦ C, under humidified 5% CO2 at­
mosphere (REVCO – CO2 incubator, Thermo Scientific, Waltham, MA,
USA) and monitored every 24 h under optical microscope (Eclipse
TS100, Nikon, Melville, New York, U.S.A.). Cultures were periodically
plated every 24 or 48 h. Cells were seeded (2 × 10 4 cells/mL) in 96-
wells and incubated for 48 h, until they reached the semi-confluence
stage. Next, Balb/c 3T3 cells were used for cytotoxicity assay. Mito­
chondrial cytotoxicity was evaluated by reducing tetrazolium salt into
formazan, which is an insoluble and colored compound. This procedure
was followed by incubation in humidified atmosphere with 3.5% CO2
(Fotakis and Timbrell, 2006).
In order to analyze the cytotoxicity of the systems, the medium was
modified by incorporation of different concentrations of free drug
(amitraz or fluazuron) and loaded (CS_PCLnp_Amitraz and
CS_PCLnp_Fluazuron) or unloaded systems (CS_PCLnp). The treatment
medium of Balb/c3T3 cells was removed after 2 h of incubation and
replaced by a medium without serum but containing MTT ([3-(4,5-
dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium] bromide) (0.5 mg/mL)
for 3 h at 37 ◦ C. This step was followed by remotion of medium and

3
E. Berni et al. Ticks and Tick-borne Diseases 13 (2022) 101849

addition of 0.1 mL of ethanol to solubilize the formazan crystals, after

agitation of plates for 10 min. The absorbance corresponding to each
well was recorded at 570 nm in UV–Vis spectrophotometer (BIO-TEK Elx
800 spectrophotometer, BioTek Instruments Inc. Winooski, VT, USA).
The data were presented as the mean (± standard deviations, n = 6)
relative to the percentage reduction of the MTT relative to the control
group (cell not exposed to any drug or nanoparticles) (Barbosa et al.,
2013; Mosmann, 1983).

2.8. Animal management

The use of animals in the current study met the requirements of the
International Guiding Principles for Biomedical Research Involving
Animals, which were developed by the Council for International Orga­
nizations of Medical Sciences (CIOMS), by following UNICAMP insti­
tutional animal care and ethics permission (Proc. 3086–1, June 3rd,
2013).

2.9. Animal studies Fig. 2. Release profile of CS_PCLnp_ Amitraz and CS_PCLnp_ Fluazuron. (PBS
with Tween (10 mg/mL).

Studies were carried out to evaluate the ability of acaricide nano­

particles to eliminate ticks from Holstein cows subjected to an acaricidal
bath. Experiments were performed on a private property located in Nova
Odessa city, São Paulo State in Brazil. The animal test was carried out
between July and August (winter, dry months in the southern
hemisphere).
The animals used were Holstein Friesian cattle, aged between 24 and
48 months, weighing between 475 and 525 kg. The average infestation
by semi-engorged female ticks was discriminated based on previously
described morphological criteria (Barker and Walker, 2014; Walker
et al., 2003). The first study comprised a 10-animal herd presenting
good nutritional status, naturally infested with R. microplus, before and
during the test.
The animals were randomly split into two groups (control and
treatment group) with five animals each. After the bath, handheld
sprinkler method, a period of 3–4 h was expected to dry the CS_PCLnp
(without any active ingredient) and CS_PCLnp_Amitraz (with amtiraz
active molecule) nanoparticles systems. The two groups (control and
treatment) remained separated in an individual paddock for each one in
an outdoor place, being exposed to natural (re)infestation of larval ticks.
Counting the number of attached female ticks per animal was carried
out, individually, in an indoor area (the corral). Each animal was
monitored for the presence of semi-engorged female ticks larger than
4.0 mm, which were counted by the same person at different times (one
day before treatment and after 1, 4, 7, 14, and 21 treatment days), based
on the Wharton and Utech method (Wharton and Utech, 1970).
The control group animals were subjected to CS_PCLnp nanoparticles
(without any active ingredient), whereas the treatment group animals
were subjected to CS_PCLnp_Amitraz. The administered amitraz dose
was based on commercial products that contain amitraz, such as Tri­
atox®. In this study, 1.25 g amitraz per animal was used in a
CS_PCLnp_Amitraz aqueous solution (125 mg/L of amitraz).
CS_PCLnp_Amitraz, obtained according to section 2.2, was diluted in
water up to 125 mg/L of the drug. The same dilution was made in
CS_PCLnp to carry on the control group test. Both aqueous solutions
CS_PCLnp and CS_PCLnp_Amitraz were applied in the animal control
and treatment group, respectively. The same farmer conducted the bath
using 10 L of the aqueous solution per animal by the product’s handheld
Fig. 3. Differential scanning calorimetry curves of nanoparticles with and
sprinkler method.
without drug, amitraz, CS_PCLnp and CS_PCLnp_Amitraz (A) and fluazuron,
The second study used two adult cows to primarily evaluate the CS_PCLnp and CS_PCLnp_Fluazuron (B).
effectiveness of a nanoparticle mix comprising amitraz and fluazuron.
Aqueous solutions comprising CS_PCLnp_Amitraz (125 mg/L) and
animal groups were observed and photographed for 28 days to monitor
CS_PCLnp_Fluazuron (62.5 mg/L) were concomitantly applied against
the effect of nano formulations regarding the presence or reduction of
ticks. The control group was subjected to the application of empty
ticks.
nanoparticles (CS_PCLnp). Qualitative analysis was carried out at
different times based on visual observation. The untreated and treated

4
E. Berni et al. Ticks and Tick-borne Diseases 13 (2022) 101849

Fig. 4. Average diameter (black close square) and zeta potential (gray open
square) of CS_PCLnp_Amitraz and (A) and CS_PCLnp_Fluazuron (B) as a function
of pH.

3. Results and discussion

Fig. 5. Average diameter (black close square) and zeta potential (gray open
Two compounds - amitraz and fluazuron - were encapsulated in the square) of CS_PCLnp_Amitraz and (A) and CS_PCLnp_Fluazuron (B) in function
current study. The main goals were to improve drug solubility, release, of NaCl concentration.
adhesion effect on hosts’ skin and penetration efficiency into the tick
parasite (Mazzarino et al., 2012). Chitosan-coated PCL nanoparticles
were manufactured and capable of loading different lipophilic com­
pounds, as well as of changing yield entrapment. The mean diameter of
unloaded nanoparticles (CS_PCLnp) was 260 ± 30 nm, whereas that of
loaded nanoparticles increased for both CS_PCLnp_Amitraz (275 ± 30
nm) and CS_PCLnp_Fluazuron (295 ± 35 nm), with PDI between 0.3 and
0.7 for all samples.
Transmission electron microscopic (TEM) images of CS_PCLnp_A­
mitraz (Fig. 1A) and CS_PCLnp_Fluazuron (Fig. 1B) showed spherical
morphology, delimited contours, and sizes in compliance with DLS and
NTA results (Fig. 1C - D). According to frozen NTA video screen images
for CS_PCLnp_Amitraz (Fig. 1E) and CS_PCLnp_Fluazuron (Fig. 1F), both
systems presented similar nanoparticle concentration; all nanoparticles
presented positive superficial charge: 40 ± 10 mV for CS_PCLnp; 43 ± 7
mV for CS_PCLnp_Amitraz; and 45 ± 10 mV for CS_PCLnp_Fluazuron.
The entrapment efficiency of the systems was different from each
other due to different interactions between the polymer PCL matrix and
Fig. 6. Dependency of nanoparticles (CS_PCLnp, CS_PCLnp_Amitraz, and
active molecules. CS_PCLnp_Amitraz had an entrapment efficiency of 77
CS_PCLnp_Fluazuron) average diameter with temperature increase.
± 1%, whereas for CS_PCLnp_Fluazuron it was 89 ± 1%. Amitraz and
Fluazuron release in vitro was slow in both nanoparticle systems.
measurements (Fig. 3A and 3B) previously taken in similar structures
CS_PCLnp_Amitraz and CS_PCLnp_Fluazuron release after 5 d was lower
(Mundargi et al., 2007).
than 20 and 30%, respectively (Fig. 2).
The stability of the evaluated systems was investigated by varying
Active compounds were molecularly dispersed (mostly) into the
pH, saline concentration, temperature, and time after production. Both
polymer PCL matrix. This outcome was confirmed by decreased melting
loaded systems showed nanoparticle agglomeration at pH higher than 7
point peak of amitraz crystals (86 ◦ C) and omission of the two melting
(Fig. 4A and 4B). Zeta potential changed with pH, as expected. Surface
point peaks from fluazuron crystals (220 ◦ C and 240 ◦ C) in the nano­
charge was positive in an acidic environment due to amine (-NH2)
structure, in comparison to the simple physical mix, as shown in DSC

5
E. Berni et al.
Fig. 7. The cytotoxicity on Balb/c 3T3 culture cells (MTT (3-[4,5-dimethylth­
iazol-2-yl]− 2,5-diphenyl tetrazolium bromide) assays): Free drugs (A),
CS_PCLnp and CS_PCLnp_Amitraz and CS_PCLnp and CS_PCLnp_Fluazuron (B).
groups of chitosan, which are more protonated (-NH3+). The number of
protonated amine groups decreased, and so did the positive surface
charge or zeta potential value, as pH changed from acidic to basic
(Fig. 4A and Fig. 4B).
The mean diameter of all systems remained stable, even at high NaCl
solution concentration (1.25 mol/L); however, zeta potential did not
remain positive with the increase of NaCl (Fig. 5A and 5B). It is worth
mentioning that none of the systems agglomerated when zeta potential
value was close to 0 mV. It may have happened due to the steric effect of
the chitosan chain (Cao and Wang, 2011).
Agglomeration of nanoparticles was also observed as temperature
increased. CS_PCLnp and CS_PCLnp_Amitraz maintained their mean di­
ameters up to 70 ◦ C, whereas CS_PCLnp_Fluazuron presented nano­
particles agglomeration at 50 ◦ C (Fig. 6). In both systems the
agglomeration nanoparticle processes were irreversible.
3.1. Cytotoxicity
Cytotoxic effect on Balb/c 3T3 cell culture was observed in the
presence of pure active ingredients (amitraz and fluazuron) and in
encapsulated systems CS_PCLnp_Amitraz and CS_PCLnp_Fluazuron
(Fig. 7A and 7B). The cytotoxic effect of unloaded nanoparticles was also
evaluated; results showed that they were safe under experimental con­
ditions of 10–200 µg/mL (cell viability higher than 90%). Amitraz and
fluazuron encapsulated in PCL nanoparticles effectively reduced the
toxicity effect on Balb/c 3T3 cells; however, cell viability gradually
decreased based on acaricide concentration loading, in both cases.
Unloaded nanoparticles did not have an effect on cell viability; however,
6
Ticks and Tick-borne Diseases 13 (2022) 101849
Fig. 8. Effect of CS_PCLnp_Amitraz against R. (B.) microplus tick based on the
average tick count of semi-engorged females. The numerical values of ticks are
presented as percentages (%), considering 100% as the value counted before the
treatment with CS_PCLnp_Amitraz. Data are presented as mean ± standard
deviation. Before*= one day before treatment.
IC50 was approximately 5 µg/mL and 100 µg/mL for pure and encap­
sulated amitraz CS_PCLnp_Amitraz, respectively (Fig. 7A). Encapsulated
amitraz thus reduced cytotoxicity. The IC50 recorded for the free and
encapsulated fluazuron forms were approximately 10 µg/mL and 30 µg/
mL, respectively (Fig. 7B), but they did not increase as much as the one
recorded for the CS_PCLnp_Amitraz system. The polymeric nanoparticles
described in the present research were excellent pesticide carriers and
have great potential for topical application in the veterinary field, since
they showed low toxicity for Balb/c 3T3 cells, mainly the
CS_PCLnp_Amitraz system.
3.2. Studies with animals
The animal tests were carried out between July and August (during
winter and dry months in the southern hemisphere) considering Kasai
et al., 2000 that showed the populational dynamics of R. microplus ticks
in Piracicaba city, localized 40 km from Nova Odessa. According to these
authors, the lowest parasitic burdens occurred during the winter months
(dry season) and the density of larval ticks naturally (re)infesting the
cows was low for both groups control and treatment, respectively in that
same period (Kasai et al., 2000).
Tick count assays were conducted in vivo one day before treatment,
and 1, 4, 7, 14, 21 days after treatment with CS_PCLnp_Amitraz (1.25 g/
animal) system on the skin of animals in order to monitor the number of
live semi-engorged female R. microplus ticks on each animal (Fig. 8).
Ticks were assumed to belong to R. microplus only after morpho­
logical microscopy analysis. For this study, we discriminated tick species
on the basis of previously described morphological criteria according to
(Barker and Walker, 2014; Roy et al., 2018; Walker et al., 2003). In
addition, according to Roy and colleagues, until March 2018, only
R. microplus had been identified in Brazil. These authors also presented
schematically the distribution of the subgenus Boophilus worldwide
based on the sequences of the cox1 gene available in GenBank (Roy
et al., 2018).
Both experimental groups had semi-engorged female ticks counted
one day before the animal’s treatment with formulations. The average
number of ticks found on animals for control and treated groups was
40.4 and 34.4, respectively (see absolute values on supplementary ma­
terial, S1). These values were considered to being 100% for each of
them.
Results show that in the naturally infested control, there was no
decline of the number of semi-engorged female ticks larger than 4.0 mm
over the 21-d observation period (Fig. 8). These results were expected
since female ticks detached during the observation period can be
E. Berni et al.
Fig. 9. Pictures of a cow treated with co-administration of CS_PCLnp_Amitraz
and CS_PCLnp_Fluazuron (inserted control untreated animal) shown the infes­
tation by R. (B.) microplus ticks. Visual observation happed for 28 days.
replaced by other female ticks and immatures on the assay’s first day.
The animals-treated group with CS_PCLnp_Amitraz showed differ­
ences between the infestation of ticks (female) before treatment (one
day before) and on subsequent days after the product application, with
more strength in 4th day. Only from the 4th day was observed signifi­
cant differences in the percentage of ticks presenting on the animal skin
comparing before and after treatment (i.e., after product applying) ac­
cording to Fig. 8. Any discrepancies were observed between the control
and treated groups during the first twenty-four hours of the experiment.
In Fig. 8, it was also possible to observe an evident decline in
R. microplus infestation after 4 d of a single bath of nano formulations
with amitraz. Also, only after 21d of the assay, it was possible to see
more attached female ticks on the treated cows’ group; it means 7
d more than the time often observed for commercial products. The
attached female ticks increase on days 7 d and 14 d, were inside the bar
error.
The results indicate improved effectiveness of the CS_PCLnp_Amitraz
system, which can help reduce costs for large cattle owners or even the
toxicity caused by prolonged use of drugs in animals. The detachment of
ticks from the cows may have happened due to amitraz action mecha­
nism, since it inhibits the monoaminoxidase enzyme and causes hyper­
arousal (Jonsson et al., 2010). This fact has suggested that the
Fig. 10. Pictures of female R. (B.) microplus in different stages before treatment (A), after the 5th day of treatment (B) with the nanosystems (CS_PCLnp_Amitraz and
CS_PCLnp_Fluazuron). (C) and (D) show exoskeleton frailty in image magnification.
7
Ticks and Tick-borne Diseases 13 (2022) 101849
CS_PCLnp_Amitraz system is significantly active against R. microplus.
The second test was only conducted with few animals because
cytotoxicity results recorded for fluazuron in 3T3 cells have shown
higher toxicity than that of amitraz (IC50 was 30 µg/mL against 100 µg/
mL). Thus, the second case study was only based on the visual analysis of
photos taken before and after the application of the associated nano­
systems. The control animal was followed-up exactly like the treated
one. Pictures of the animals’ necks were taken after nanosystems
application (Fig. 9). Decline in infestation by R. microplus ticks was only
observed after the 4th experimental day; however, between the 5–9th
days after bathing with nanosystems association (CS_PCLnp_Amitraz
and CS_PCLnp_Fluazuron), there was a distinct decline in tick infesta­
tion. Just like the first test, the knock down effect (detachment effect)
was attributed to amitraz molecules (Prullage et al., 2011).
Exoskeleton frailty could be noticed at the time ticks were removed,
some of them popped due to chitosan lack in the exoskeleton (Fig 10B).
Interestingly, dry ticks were rigidly attached to cowhide (Fig. 10C and
10D), since it was possible seeing cowhide pieces trapped in the
mouthparts of R. microplus.
4. Final remarks
The current results showed that lipophilic active molecules did not
change the positive charge of nanoparticles after CS adsorption on
nanoparticles surface, confirming that active molecules are dispersed in
the PCL polymer matrix. Moreover, loading capacity and entrapment
efficiency directly depended on the active molecules used. The
CS_PCLnp_Fluazuron system showed loading capacity of 22 ± 1 (mg/
100 mg active/nanoparticles) and entrapment efficiency of 89 ± 1%.
CS_PCLnp_Amitraz performed better than the other system since it
showed a loading capacity of 39 ± 1 (mg/100 mg active/nanoparticles)
and entrapment efficiency of 77 ± 1%. Both active molecules were
dispersed in the matrix. These systems decreased the toxicity of the free
active drug acting on 3T3 culture cells. The most promising outcome
was the positive bovine protection effect against R. microplus in vivo.
Therefore, these polymeric nanoparticles have great potential to protect
cattle against ticks.
CRediT authorship contribution statement
Elias Berni: Conceptualization, Methodology, Formal analysis,
Investigation, Resources, Writing – original draft. Raquel de Melo
Barbosa: Methodology, Formal analysis, Investigation, Writing –
E. Berni et al.
original draft, Writing – review & editing. Nelson Durán: Writing –
review & editing, Supervision, Project administration, Funding
acquisition.
Declaration of Competing Interests
The authors declare that this research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Funding
This research was financially supported by FAPESP and CNPq.
Acknowledgements
We acknowledge the support from the São Paulo Research Founda­
tion (FAPESP), the National Council for Scientific and Technological
Development (CNPq), the Ministry of Science, Technology, Innovation
and Communications (MCTI), the National Institute of Science and
Technology in Functional Complex Materials (INOMAT) (MCTI/CNPq),
the Oncology Nanotoxicology Group NanoBioss (MCTI) and the Brazil­
ian Network in Nanotoxicology (CIGENANOTOX). We also appreciate
the initial collaboration with Dr. P.D. Marcato, during her post-
doctorate in our institution.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ttbdis.2021.101849.
References
Akre, C., 2017. The Use of Pyrethroids, Carbamates, Organophosphates, and Other
Pesticides in Veterinary Medicine. Chemical Analysis of Non-Antimicrobial
Veterinary Drug Residues in Food. JohnWiley & Sons, Inc., Hoboken, New Jersey,
pp. 383–426. https://doi.org/10.1002/9781118696781.ch7.
Andreotti, R., Koller, W.W., Garcia, M.V., 2016. Carrapatos: Protocolos e Técnicas Para
Estudo, I. ed. Embrapa, Brasilia.
Ángel-sahagún, C.A., Guanajuato, U.De, Lezama-gutiérrez, R., Molina-ochoa, J., 2010.
Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales:
clavicipitaceae) upon Rhipicephalus = Boophilus microplus (Acari: ixodidae) larvae
and the efficacy of conidia formulations to reduce larval tick density under field
conditions. Vet. Parasitol. 170, 278–286. https://doi.org/10.1016/j.
vetpar.2010.02.037.
Barbosa, R.M., Da Silva, C.M.G., Bella, T.S., De Araújo, D.R., Marcato, P.D., Durán, N.,
De, Paula, E., 2013. Cytotoxicity of solid lipid nanoparticles and nanostructured lipid
carriers containing the local anesthetic dibucaine designed for topical application.
J. Phys. Conf. Ser. 429 https://doi.org/10.1088/1742-6596/429/1/012035.
Barker, S., Walker, A.R., 2014. Ticks of Australia. The species That Infest Domestic
Animals and humans, Monograph. Magnolia Press, Auckland. https://doi.org/
10.11646/zootaxa.3816.1.1. Zootaxa.
Behrenz, W., Schutte, M., 1989. Synergistic pesticidal composition. US 4863909.
Beltrão, M., Silva, F., Buzatti, A., Staude, F., Kan, L., Coimbra, E., Dorneles, L., 2013.
Partial selective treatment of Rhipicephalus microplus and breed resistance variation
in beef cows in Rio Grande do Sul, Brazil. Vet. Parasitol. 192, 234–239. https://doi.
org/10.1016/j.vetpar.2012.10.021.
Bouvier, J., Kolly, C., 2005. Combination of fluazuron and ivermectin for parasite
control. EP 1 244 359 B1.
Brasil, MAPA, 2012. INSTRUÇÃO NORMATIVA No 15, DE 9 DE MAIO DE 2005.
Legislação Relacionada Aos Produtos De Uso Veterinário, 1st ed. MAPA/ACS,
Brasília.
Calvano, M.P.C.A., Brumatti, R.C., Garcia, M.V., Barros, J.C., Andreotti, R., 2019.
Economic efciency of Rhipicephalus microplus control and efect on beef cattle
performance in the Brazilian Cerrado. Exp. Appl. Acarol. 79, 459–471. https://doi.
org/10.1007/s10493-019-00446-5.
Cao, G., Wang, Y., 2011. Nanostructures & nanomaterials: synthesis, properties, and
applications, 2nd ed. World Scientific, Nanostructures & nanomaterials: synthesis,
properties, and Applications, New Jersey. https://doi.org/https://doi.org/10.1142/
7885.
Dantas-Torres, F., Chomel, B.B., Otranto, D., 2012. Ticks and tick-borne diseases: a One
Health perspective. Trends Parasitol 28, 437–446. https://doi.org/10.1016/j.
pt.2012.07.003.
Dos Santos, T.R.B., Pappen, F.G., Farias, N.A.R., Vaz Junior, I.S., 2009. In vitro analysis of
amitraz efficacy against Rhipicephalus (Boophilus) microplus (Canestrini, 1887)
8
Ticks and Tick-borne Diseases 13 (2022) 101849
populations of southern region of Rio Grande do Sul state. Rev. Bras. Parasitol. Vet.
18, 54–57. https://doi.org/10.4322/rbpv.018e1010.
Duran, N., Berni Neto, E.A., Marcato Gaspari, P.D., Sabatini, G., 2014. Polymer
nanoparticles containing amitraz and/or fluazuron, production method, formulation
and uses. WO 2014/094087 Al.
Ferreira, C., Almeida, T., Barbosa, R.D.M., Cordeiro, J., Morsink, M., Souto, E.B.,
Severino, P., 2020. New Trends in Drug Delivery Systems for Veterinary
Applications. Pharm. Nanotechnol. 55, 1–16. https://doi.org/10.2174/
2211738508666200613214548.
Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S., 1989. Nanocapsule
formation by interfacial polymer deposition following solvent displacement. Int. J.
Pharm. 55, R1–R4. https://doi.org/10.1016/0378-5173(89)90281-0.
Filipe, V., Hawe, A., Jiskoot, W., 2010. Critical evaluation of Nanoparticle Tracking
Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein
aggregates. Pharm. Res. 27, 796–810. https://doi.org/10.1007/s11095-010-0073-2.
Filippo, P.A.Di, Sousa, R.V., Vieras, L.Q., Santana, G.C., Abrahamsohn, I.A., Oliveira, M.
A.P., 2006. Anti-inflammatory effect of amitraz due to inhibition of the gamma-
interferon production and mitochondrial activity of the T lymphocytes. ARS Vet 22,
138–145. https://doi.org/10.15361/2175-0106.2006v22n2p138-145.
Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assay: comparison of LDH, neutral
red, MTT and protein assay in hepatoma cell lines following exposure to cadmium
chloride. Toxicol. Lett. 160, 171–177. https://doi.org/10.1016/j.
toxlet.2005.07.001.
Gonçalves, S., Hugo, V., Araujo, S., Martins, A., Lobato, J., Letícia, A., Silvestre, P.,
Fonseca-santos, B., Cecília, J., Villanova, O., Palmira, M., Chorilli, M., 2020.
Advances and challenges in nanocarriers and nanomedicines for veterinary
application. Int. J. Pharm. 580, 1–15. https://doi.org/10.1016/j.
ijpharm.2020.119214.
Grisi, L., Leite, R.C., Martins, J.R.de S., Barros, Antonio Thadeu Medeiros de,
Andreotti, R., Cançado, Paulo Henrique Duarte, León, A.A.P.de, Pereira, Jairo
Barros, Villela, H.S., 2014. Reassessment of the potential economic impact of cattle
parasites in Brazil. Braz. J. Vet. Parasitol., Jaboticabal 23, 150–156. https://doi.org/
10.1590/S1984-29612014042.
Grisi, L., Massard, C.L., Moya-Borja, G.E., Pereira, J.B., 2002. Impacto econômico das
principais ectoparasitoses em bovinos no Brasil. A Hora Veterinária 21 8–10.
Hunter III, J.S., Baggott, D., Everett, W.R., Fourie, J.J., Cramer, L.G., Yoon, S.S.,
Collidor, N., Mallouk, Y., Lee, L., Blair, J., Prullage, J.B., 2011. Efficacy of a novel
topical combination of fipronil, amitraz and (S)-methoprene for treatment and
control of induced infestations of brown dog ticks (Rhipicephalus sanguineus) on dogs.
Vet. Parasitoogy 179, 318–323. https://doi.org/10.1016/j.vetpar.2011.03.042.
Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129,
S3–S14. https://doi.org/10.1017/S0031182004005967.
Jonsson, N.N., Miller, R.J., Kemp, D.H., Knowles, A., Ardila, A.E., Verrall, R.G.,
Rothwell, J.T., 2010. Rotation of treatments between spinosad and amitraz for the
control of Rhipicephalus (Boophilus) microplus populations with amitraz resistance.
Vet. Parasitol. 169, 157–164. https://doi.org/10.1016/j.vetpar.2009.12.026.
Kasai, N., Labruna, M.B., Pires, A.V., Louvandini, H., Abdalla, A.L., Gennari, S.G., 2000.
Populational dynamics of Boophilus microplus (Canestrini, 1887) in dairy cattle under
intensively grazing elephant grass pasture. Arq. Bras. Med. Veterinária e Zootec. 52,
453–458. https://doi.org/10.1590/S0102-09352000000500008.
Kaushik, A., 2019. Biomedical Nanotechnology Related Grand Challenges and
Perspectives. Front. Nanotechnol. 1, 1–4. https://doi.org/10.3389/
fnano.2019.00001.
Kryger, U., Deschodt, C., Davis, A.L.V, Scholtz, C.H., 2007. Effects of cattle treatment
with a fluazuron pour-on on survival and reproduction of the dung beetle species
Onthophagus gazella (Fabricius). Vet. Parasitol. 143, 380–384. https://doi.org/
10.1016/j.vetpar.2006.08.021.
Kumar, S., Nehra, M., Dilbaghi, N., Marrazza, G., Aly, A., Kim, K., 2019. Nano-based
smart pesticide formulations: emerging opportunities for agriculture. J. Control.
Release 294, 131–153. https://doi.org/10.1016/j.jconrel.2018.12.012.
Madder, M., Thys, E., Achi, L., Touré, A., Deken, R.De, 2011. Rhipicephalus (Boophilus)
microplus: a most successful invasive tick species in West-Africa. Exp. Appl. Acarol.
53, 139–145. https://doi.org/10.1007/s10493-010-9390-8.
Marcato, P.D., Durán, N., 2008. New Aspects of Nanopharmaceutical Delivery Systems.
J. Nanosci. Nanotechnol. 8, 2216–2229. https://doi.org/10.1166/jnn.2008.274.
Matzembacker, B., Collet, S.G., Prestes, A.M., Camillo, G., 2019. Factors associated with
the efficiency of acaricides on different populations of Rhipicephalus microplus. Braz
J. Vet. Res. Anim. Sci. 56, 1–7 https://doi.org/issn.1678-4456.bjvras.2019.157595.
Mazzarino, L., Travelet, C., Ortega-murillo, S., Otsuka, I., Pignot-paintrand, I., Lemos-
senna, E., Borsali, R., 2012. Elaboration of chitosan-coated nanoparticles loaded
with curcumin for mucoadhesive applications. J. Colloid Interface Sci. 370, 58–66.
https://doi.org/10.1016/j.jcis.2011.12.063.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
https://doi.org/10.1016/0022-1759(83)90303-4.
Mundargi, R.C., Srirangarajan, S., Agnihotri, S.A., Patil, S.A., 2007. Development and
evaluation of novel biodegradable microspheres based on poly (D, L-lactide-co-
glycolide) and poly (ε-caprolactone) for controlled delivery of doxycycline in the
treatment of human periodontal pocket: in vitro and in vivo studies. J. Control.
Release 119, 59–68. https://doi.org/10.1016/j.jconrel.2007.01.008.
Patra, J.K., Das, G., Fraceto, L.F., Vangelie, E., Campos, R., Rodriguez, P., Susana, L.,
Torres, A., Armando, L., Torres, D., Grillo, R., 2018. Nano based drug delivery
systems: recent developments and future prospects. J. Nanobiotechnology 16, 1–33.
https://doi.org/10.1186/s12951-018-0392-8.
Periyannan, R., Prabhu, N.M., Ramasamy, P., Murugan, K., Benelli, G., 2017.
Exploitation of chemical, herbal and nanoformulated acaricides to control the cattle
E. Berni et al.
tick, Rhipicephalus (Boophilus) microplus- A review. Vet. Parasitol. 244, 102–110.
https://doi.org/10.1016/j.vetpar.2017.07.021.
Plimmer, J.R., Gammon, D.W., Ragsdale, N.N., 2003. Encyclopedia of Agrochemicals, 3.
Wiley-Interscience, New York illustrate. ed.
Pourseyed, S.H., Tavassoli, M., Bernousi, I., Mardani, K., 2010. Metarhizium anisopliae
(Ascomycota: hypocreales): an effective alternative to chemical acaricides against
different developmental stages of fowl tick Argas persicus (Acari: argasidae). Vet.
Parasitol. 172, 305–310. https://doi.org/10.1016/j.vetpar.2010.05.014.
Prullage, J.B., Tran, H.V., Timmons, P., Harriman, J., Chester, S.T., Powell, K., 2011. The
combined mode of action of fipronil and amitraz on the motility of Rhipicephalus
sanguineus. Vet. Parasitol. 179, 302–310. https://doi.org/10.1016/j.
vetpar.2011.03.041.
Ren, Q., Liu, Z., Guan, G., Sun, M., Ma, M., Niu, Q., Li, Y., Liu, A., Liu, J., Yang, J.,
Yin, H., Luo, J., 2012. Laboratory evaluation of virulence of Chinese Beauveria
bassiana and Metarhizium anisopliae isolates to engorged female Rhipicephalus
(Boophilus) microplus ticks. Biol. Control 63, 98–101. https://doi.org/10.1016/j.
biocontrol.2012.07.002.
Rosa, P., Oliveira, D., Braggião, I., Cristina, G., Henrique, G., Aparecido, M., Izabel, M.,
Mathias, C., 2012. Potential of the insect growth regulator, fluazuron, in the control
of Rhipicephalus sanguineus nymphs (Latreille, 1806) (Acari: ixodidae): determination
Ticks and Tick-borne Diseases 13 (2022) 101849
of the LD95 and LD50. Exp. Parasitol. 131, 35–39. https://doi.org/10.1016/j.
exppara.2012.02.023.
Roy, B.C., Estrada-peña, A., Krücken, J., Rehman, A., 2018. Morphological and
phylogenetic analyses of Rhipicephalus microplus ticks from Bangladesh, Pakistan and
Myanmar. Ticks Tick-borne Dis. J. 9, 1069–1079. https://doi.org/10.1016/j.
ttbdis.2018.03.035.
Thiruvengadam, M., Rajakumar, G., Min, I., 2017. Nanotechnology: current uses and
future applications in the food industry. J. Food Drug Anal. 25, 245–253. https://
doi.org/10.1007/s13205-018-1104-7.
Walker, A.R., 2011. Eradication and control of livestock ticks: biological, economic and
social perspectives. Parasitology 138, 945–959. https://doi.org/10.1017/
S0031182011000709.
Walker, A.R., Bouattour, A., Horak, I., 2003. Ticks of Domestic Animals in Africa: a Guide
to Identification of Species. Bioscience Reports, Edinburgh, Scotland,UK.
Wharton, R.H., Utech, K.B.W., 1970. The relation between engorgement and dropping of
Boophilus microplus (Canestrini) (lxodidae) to the assessment of tick numbers on
cattle. J. Aust. Entomol. Soc. 9, 171–182. https://doi.org/10.1111/j.1440-
6055.1970.tb00788.x.
Wu, X., Na, R., Wei, S., Zhu, J., Peng, H., 2013. Distribution of tick-borne diseases in
China. Parasit. Vectors 6, 1–8. https://doi.org/10.1186/1756-3305-6-119.