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Article

Thymol-Loaded Polymeric Nanocapsules’ Repellent Activity on Nymphs of Rhipicephalus sanguineus Sensu Lato

by
Amanda M. R. Sales
1,
Gessyka R. S. Pereira
2,
Lais C. N. Lima
2,
Caio M. O. Monteiro
3,
Breno N. Matos
1,
Stephânia F. Taveira
2,
Marcilio Cunha-Filho
1,
Guilherme M. Gelfuso
1 and
Tais Gratieri
1,*
1
Laboratory of Food, Drugs, and Cosmetics (LTMAC), University of Brasilia, Brasilia 70910-900, DF, Brazil
2
Laboratory of Nanosystems and Drug Delivery Devices (NanoSYS), Faculdade de Farmácia, Universidade Federal de Goiás (UFG), Rua 240, Setor Leste Universitário, Goiânia 74605-170, GO, Brazil
3
Laboratório de Biologia, Ecologia e Controle de Carrapatos (LaBEC), Instituto de Patologia Tropical e Saúde Pública da Universidade Federal de Goiás (UFG), Rua 235, Setor Universitário, Goiânia 74605-050, GO, Brazil
*
Author to whom correspondence should be addressed.
Coatings 2024, 14(10), 1295; https://doi.org/10.3390/coatings14101295
Submission received: 16 September 2024 / Revised: 3 October 2024 / Accepted: 9 October 2024 / Published: 11 October 2024
(This article belongs to the Special Issue Organic and Polymeric Materials: Synthesis, Properties and Applications)
Browse Figures
Figure 1
<p>NPTML stability. The samples were stored at 4 °C and 40 °C, and the parameters of (<b>A</b>) hydrodynamic diameter, (<b>B</b>) polydispersity index <span class="html-italic">(PdI)</span>, (<b>C</b>) zeta potential, (<b>D</b>) pH, (<b>E</b>) encapsulation efficiency, and (<b>F</b>) drug content were evaluated for 90 days (n = 3).</p> "> Figure 2
<p>TEM images of thymol-loaded polymeric nanocapsules at different magnifications.</p> "> Figure 3
<p>Thymol release profile from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control over 24 h (n = 5).</p> "> Figure 4
<p>Thymol skin delivery from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control. After 2 h of skin treatment, thymol was recovered from the stratum corneum (SC), hair follicle (HF), and remaining skin (RS) (n = 5). ** <span class="html-italic">p</span> &lt; 0.01.</p> "> Figure 5
<p>First derivate of the TGA analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.</p> "> Figure 6
<p>DSC analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.</p> ">
Versions Notes

Abstract

:
Thymol-loaded polymeric nanocapsules were developed in this study to control volatilization and drug release for repellent application on Rhipicephalus sanguineus nymphs. Policaprolactone-loaded nanocapsules were prepared and characterized by diameter, PdI, zeta potential, pH, entrapment efficiency, and thymol content. Moreover, drug release, skin permeation profile, and repellent activity were evaluated. Nanocapsules showed a mean diameter of 195.7 ± 0.5 nm, a PdI of 0.20 ± 0.01, a zeta potential of −20.6 ± 0.3 mV, a pH of 4.7 ± 0.1, and an entrapment efficiency and a thymol content of 80.1 ± 0.1% and 97.9 ± 0.2%, respectively. The nanosystem progressively released 68.6 ± 2.3% of the thymol over 24 h, demonstrating that it can control drug release. Thymol-loaded nanocapsules showed less epidermis penetration upon skin application than pure thymol (control). Moreover, nanocapsules showed 60–70% repellency for 2 h against Rhipicephalus sanguineus nymphs. Thus, the nanocapsules proved to be a promising alternative for use as an arthropod repellent.

1. Introduction

Topical repellents play an essential role in personal protection. The topical use of N, N-diethyl-m-toluamide (DEET) is recommended as the gold standard against blood-sucking vectors, such as mosquitos and ticks [1,2,3,4]. However, synthetic repellents are being questioned for their safety, especially for pregnant women, children, and babies. In addition, the cosmetic characteristics, such as oil consistency and pungent odor, may lead to low usage of these topical products [5,6,7].
Thus, natural-based repellents have been studied as an alternative to synthetic repellents. Thymol (2-isopropyl-5-methylphenol) is a natural molecule known for its instability under oxygen, light, and temperature and for its low aqueous solubility, which hampers its incorporation into topical formulations [5,8]. Among a wide range of applications such as an antioxidant, anti-inflammatory, antibacterial, and anticarcinogenesis [9,10,11] agent, thymol is particularly interesting as a repellent and as a form of pest control [12,13]. Indeed, thymol presents acaricide activity on Amblyomma cajennense, Rhipicephalus sanguineus, and Rhipicephalus turanicus [14] and repellent activity on Rhipicephalus (Boophilus) microplus [15,16,17].
Despite its physical instability, some authors have reported that natural substances provide protection for a short time [18,19]. Thus, the encapsulation of hydrophobic drugs such as thymol in biodegradable polymeric nanoparticles has been described as a promising delivery system that can improve their solubility, stability, and bioavailability; protect from toxicity and physical and chemical degradation; and sustain the delivery of bioactive molecules [20,21,22,23,24].
This present study aimed to develop thymol-loaded polymeric nanoparticles capable of preventing thymol volatility and controlling its release. Moreover, this study aimed to evaluate thymol-loaded polymeric nanoparticle repellency against blood-sucking vectors. Nymphs of Rhipicephalus sanguineus sensu lato were used to assess the formulation repellency. Also known as the brown dog tick, R. sanguineus has worldwide distribution. Although its favorite host is a dog, the R. sanguineus tick can parasitize humans and transmit pathogens to both dogs (Babesia sp. and Ehrlichia canis) and humans (Rickettsia rickettsii) [25,26].

2. Materials and Methods

2.1. Material

Synthetic thymol (Lot #SZBF0370V, ≥99% purity), 2-hydroxypropyl-β-cyclodextrin (HPβCD), sorbitan monostearate (Span 60®), poly (ε-caprolactone) (PCL), and filter paper (Whatman® Qualitative Cat No. 1001, 90 mm) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Polysorbate 80 and caprylic/capric triglyceride were purchased from Dinâmica Química Contemporânea (São Paulo, Brazil). Soybean lecithin (Lipoid S75) was obtained from Lipoid GmbH (Ludwigshafen-am-Rhein, Germany). Ethanol (99.8% purity) was obtained from ACS Científica (São Paulo, Brazil). Other solvents and chemicals were of analytical grade. All aqueous solutions were prepared with ultrapure water (Mili-Q®, Millipore, Illkirch-Graffenstaden, France).
Cellulose acetate membrane (MWCO 12,000 to 4000 Da) used in the in vitro release tests was obtained from Sigma-Aldrich (St. Louis, MO, USA). Porcine ears used in the in vitro skin permeation tests were obtained from a local abattoir (Bonasa S/A, Formosa, Brazil) from animals that were destined for meat consumption. The skin was also purchased; however, the skin was removed before the scalding process. Since the animals were not slaughtered for research purposes and because all the local legal and health requirements for operation were followed, there was no need for ethical consent.

2.2. Preparation of Polymeric Nanocapsules

Thymol-loaded nanocapsule (NPTML) dispersions were prepared by nanoprecipitation using the interfacial deposition of preformed polymers method [27] with some modifications [28]. Thymol and HPβCD were added in equimolar proportion [29]. For the organic phase, thymol (0.06%), PCL (0.25%), sorbitan monostearate (0.10%), and caprylic triglyceride (0.30%) were solubilized in acetone (25 mL). In addition, an ethanolic solution containing HPβCD (0.60%) and soybean lecithin (0.08%) was prepared (5 mL). The organic phase, obtained by mixing both solvents, was then slowly poured into an aqueous solution (50 mL) containing polysorbate 80 (0.20%) under magnetic stirring (400 rpm). Finally, the sample was concentrated using a rotaevaporator (43 mbar and 35 °C). The percentage of the components was calculated from the final volume, which was 40 mL.

2.3. Stability Studies

NPTML dispersions were stored at 4 °C and 40 °C in closed amber glasses, and the parameters of hydrodynamic diameter, polydispersity index, zeta potential, pH, encapsulation efficiency, and drug content were evaluated for 90 days.

2.3.1. Particle Size, Polydispersity Index, and Zeta Potential

The samples were diluted (1:100 v/v) using ultrapure water. Particle size, polydispersity index (PdI), and zeta potential were measured by dynamic light scattering using a Zetasizer Nanoseries (Malvern Instruments, Worcestershire, UK).

2.3.2. pH Measurements

The pH of the NPTML dispersions was measured without previous dilution using a DM-22 pH meter (Digimed Analítica Ltd., São Paulo, Brazil) at 25 °C. All samples were analyzed in triplicate.

2.3.3. Encapsulation Efficiency and Drug Content

The total thymol content (TC) in the nanocapsules was determined by mixing the NPTML dispersions (0.1 mL) with methanol (9.9 mL) on a vortex for 1 min and quantifying the thymol. The amount of the drug not entrapped (FF) was obtained by quantifying the thymol in the filtered fraction by ultrafiltration. For this, 1 mL of the nanocapsule dispersions was centrifuged using Vivaspin 2 MWCO 1.000 (Sartorius AG, Göttingen, Germany) for 20 min at 3000 rpm using a K14-4000 centrifuge (Kasvi Ltd., São José dos Pinhais, Brazil). Drug content (TC%) is the remaining amount of drug after a time, calculated by comparing TC after × days (TCdayx) compared with TC on the sample preparation day (TC). Encapsulation efficiency (EE) and drug content (DC) were calculated using Equations (1) and (2), respectively, where
E E % = T C − F F T C × 100
and
T C % = T C d a y x T C × 100 .

2.4. Nanocapsule Morphology

A drop of the NPTMLs diluted with ultrapure water (1:20 v/v) was placed on a copper grid and left to air-dry overnight at 25 °C. Then, the sample was stained with 1% phosphotungstic acid. The morphology of the polymeric nanocapsules was assessed by transmission electron microscopy (TEM). The microscope used in the experiment was a JEM-2100 at 200 kV (JEOL Inc., Boston, MA, USA).

2.5. Drug Release

An in vitro drug release study was carried out using modified Franz-type diffusion cells. The receptor compartment was filled with 0.5% w/v sodium dodecyl sulfate (SDS) (15 mL) to ensure sink conditions, as previously reported [30,31]. The dialysis membrane was mounted between the donor and the receptor compartment. NPTMLs or a control solution consisting of thymol in the same concentration used in the nanocapsules was placed into the donor compartment with 0.5% SDS. The system was maintained under stirring (500 rpm) at 37 ± 2 °C. Aliquots (1 mL) of the receptor medium were removed each hour for 12 h or for 24 h. The exact amount of the medium that was removed was replaced immediately. The amount of thymol released in each sample was determined by HPLC.

2.6. In Vitro Skin Permeation

Skin permeation of thymol-loaded nanocapsules was investigated using porcine ear skin. The skin debris was carefully removed from the outer part of the ears using a surgical scalpel. The excised skin was mounted on modified Franz-type diffusion cells. The receptor chamber was filled with 15 mL of a solution containing a phosphate buffer (pH 7.4) and 0.5% SDS. The NPTML dispersions or a control solution, in addition to thymol with ultrapure water, were placed into the donor compartment that was in contact with the stratum corneum for 2 h. Permeation experiments were maintained by stirring (500 rpm) at 37 ± 2 °C. After that, a differential tape-stripping technique was performed on the skin [32]. Thymol recovery from the stratum corneum, hair follicle, and remaining skin was carried out using 5 mL of methanol and overnight stirring (500 rpm). The samples were filtered and analyzed following the HPLC method that was previously described.

2.7. Thermal Analyses

The thermal properties were analyzed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) under a nitrogen-controlled atmosphere with a flow rate of 50 mL/min and a heating rate of 10 °C/min. Approximately 3 ± 0.1 mg of lyophilized NPTMLs, their physical mixture, and the individual components were weighted to the TGA and DSC analysis, which were performed with DTG-60 and DSC-60 (Shimadzu Corp., Kyoto, Japan), respectively. Thermal measurements were determined using the TA-60 Shimadzu software.

2.8. Repellent Activity

2.8.1. Tick Colony

Engorged females of R. sanguineus were obtained from naturally infested dogs at the Escola de Veterinária e Zootecnia (EVZ) of the Universidade Federal de Goiás (UFG) (Goiânia, Brazil). For egg posture, the engorged females were incubated at 27 ± 1 °C and >80% relative humidity. Larvae and nymphs were incubated at the same conditions described above, and rabbits (Oryctolagus cuniculus California × New Zealand) were used for feeding these stages by way of a glued feeding apparatus on the animal’s shaved back [33]. This work was approved by the Ethics Committee on Animal Use of the UFG (CEUA/UFG) (Protocol n. 058/20). Unfed nymphs aged between 30 and 60 days were used for all repellence bioassays.

2.8.2. Repellent Bioassay

Thymol-loaded nanoparticles were evaluated for repellency efficacy based on the studies by [34,35]. The experiments were performed in an acclimatized room (25 ± 1 °C) with a relative humidity of 70 ± 10%. Two semicircles of filter paper were used to cover the bottom of the Petri dish (10 cm diameter). One semicircle received 200 μL of thymol-loaded nanoparticles and the other semicircle received the same volume of ethanol (control side). Unloaded formulations were also evaluated. The filter papers were air-dried for 10 min. Then, ten unfed nymphs were placed in the center of the Petri dish between the treated and untreated papers. The Petri dishes were covered tightly with a transparent fine mesh fabric (100% polyamide) that was secured with rubber bands to prevent the nymphs from escaping. During the evaluations, ticks that remained in the untreated semicircle (control side) were considered repelled. The mean repellency percentage corresponded to the number of ticks on the control side of the Petri dish (n = 10).

2.9. Determination of Thymol

The thymol quantification analyses were processed on a reversed-phase, high-performance liquid chromatography (HPLC) with ultraviolet detection. Thymol was quantified on LC-20AT equipment (Shimadzu Corp., Kyoto, Japan) using a column RP-C18 (300 mm × 3.9 mm; 10 μm) with a volume of 30 µL and settled at 40 °C. The mobile phase was acetonitrile and water (65:35 v/v) with a flow rate of 1.5 mL/min and detection at 278 nm. Limits of detection and quantification were 0.05 μg/mL and 0.14 μg/mL, respectively [36].

2.10. Statistical Analyses

Data were analyzed using GraphPad Prism software version 8.4.2 (GraphPad Software Inc., San Diego, CA, USA). Because the data presented as a normal distribution, the means of treatments were compared using the two-way ANOVA test. Differences were considered significant if p < 0.05. A post hoc Sidak test was applied if significant differences were detected. The results are presented as mean ± standard deviation (SD). Repellent bioassay data were statistically treated with the chi-square test (p < 0.05) using the online platform quantpsy.org [36].

3. Results

3.1. Stability Studies

Figure 1 shows the stability study results of the nanocapsule samples stored over 90 days at 4 °C and 40 °C.
The PCL nanocapsules prepared by nanoprecipitation had a diameter of 195.7 ± 0.5 nm on the first day of analyses (D0). Samples stored at 4 °C were stable and no difference in their particle size occurred (p < 0.05) during the 90 days of analysis. However, the samples stored at 40 °C changed over the analyses.
Negative zeta potential values of −20.6 ± 0.4 mV were observed on D0 (Figure 1), and the pH was 4.7 at D0. A statistical difference was observed only in the sample stored at 40 °C, which saw its pH decrease to 3.8 on the 90th day. Meanwhile, the sample stored at 4 °C maintained its pH value.
The entrapment efficiency of thymol in the nanocapsules was 80.1 ± 0.08% at D0. No difference was observed for the sample at 4 °C (p > 0.05). However, the sample at 40 °C showed a decrease in entrapment efficiency and there was a statistical difference between the analyzed days. The drug content was about 97% at D0. Both samples presented statistical differences (p < 0.05). The thymol content of the sample at 40 °C decreased by more than 70% on the 90th day of the stability study.

3.2. Nanocapsule Morphology

The TEM image of NPTMLs showed nanocapsules with a spherical shape, a lighter core region, and a darker wall compartment (Figure 2).

3.3. In Vitro Release Study

The control sample released 606.7 µg of thymol in 24 h, which corresponded to 97.1 ± 1.5% of the total thymol content. During the same period, the NPTMLs released 428.7 µg of thymol, corresponding to 68.6% ± 2.3 of the thymol content. There was a statistical difference (p < 0.05) between the NPTMLs and the control for all analyzed times (Figure 3).

3.4. In Vitro Skin Permeation

Figure 4 shows the results from the permeation studies of thymol from the nanocapsules performed on porcine skin.
The control sample had a higher thymol penetration into the remaining skin compared with NPTMLs, showing penetration rates of 13.2 ± 2.3 μg/cm2 into the stratum corneum, 3.5 ± 0.8 μg/cm2 into the hair follicles, and 90.3 ± 7.6 μg/cm2 into the remaining skin. On the other hand, NPTMLs showed penetration rates of 2.0 ± 1.2 μg/cm2 into the stratum corneum, 1.0 ± 0.7 μg/cm2 into the hair follicles, and 6.7 ± 1.3 μg/cm2 into the remaining skin.
It was observed that NPTMLs increased thymol retention by three times into the hair follicles compared with the control, showing a thymol retention targeting ability. NPTMLs were capable of protecting the skin from thymol penetration, which is advantageous since active penetration into the skin is not preferable for a repellent formulation. It is expected that the retained thymol serves as an active system of retention, prolonging repellency time.

3.5. Thermal Analyses

3.5.1. TGA Analysis

The results of the TGA analyses are displayed in Figure 5.
Pure thymol showed a single mass loss initiating at 50 °C and finishing at approximately 180 °C. The physical mixture showed a mass loss of approximately 60% between 0 and 250 °C, possibly due to HPβCD water loss and thymol evaporation. Together, HPβCD and thymol represented 41% of the physical mixture.
The PCL started a slow degradation process after 300 °C and rapidly arrived at a decomposition temperature of 410 °C.

3.5.2. DSC Analysis

The results of the DSC analyses are shown in Figure 6.
Pure thymol showed an endothermic peak at 50.5 °C, corresponding to the phase transitions until evaporation. The PCL showed an endothermic peak at 57.1 °C, which was related to dehydration. HP-β-CD presented a significant endothermic event, corresponding to water loss from the hydrophobic cavity.

3.6. Repellent Bioassay

Table 1 demonstrates the percentage of repellence of thymol-loaded nanoparticles, thymol ethanolic solution, and negative controls (unloaded nanoparticles and ethanol).
Thymol-loaded nanoparticles presented a repellency of about 60–70% for 2 h, significantly differing from the negative controls (ethanol and unloaded nanoparticles) (p < 0.05). In addition, the thymol ethanolic solution presented a similar repellency percentage (p > 0.05). Thus, polymeric nanoparticles did not affect the intrinsic thymol effectiveness, providing nymph repellency for 2 h.
In summary, the present polymeric nanocapsules controlled thymol release more than the other samples used on the repellent bioassay. As shown previously, in 24 h, NPTMLs released 68.59% of the thymol. Even with controlled release, the polymeric nanostructure did not affect its repellent activity against R. sanguineus nymphs, achieving excellent repellency values (60–70%) for 2 h. These results offer interesting insights into a repellent application for topical use. A stronger repellency can be achieved by increasing the thymol concentration, which can be done by concentrating the formulation or increasing the application area.

4. Discussion

The PdI of both samples remained within a narrow range of approximately 0.2, indicating high homogeneity among the prepared nanocapsules [37]. Nanoparticles exhibiting a zeta potential greater than ±30 mV are considered stable, as their electric charge reduces the likelihood of aggregation, thereby enhancing overall stability [38]. The zeta potential, which reflects the electric potential of nanoparticles, is significantly influenced by the excipients employed during formulation. This observation aligns with findings from previous studies [39]. The negative zeta potential observed is attributable to the structural characteristics of polysorbate 80 [40] and the presence of phosphatidic acid in lecithin [41]. A decrease in zeta potential was noted in samples stored at 4 °C and 40 °C, with both samples exhibiting statistically significant differences across the analyzed days.
The pH level is a critical determinant of the stability of a formulation and its suitability for topical application, which generally requires a slightly acidic pH [42]. The pH maintenance in samples stored at 4 °C suggests no significant degradation of the PCL under these conditions, indicating that thymol remained encapsulated throughout the stability studies [43]. The observed pH decreases in samples stored at 40 °C can be attributed to PCL hydrolysis and the auto-oxidation of polysorbate 80. PCL hydrolysis results in the release of carboxylic groups, which lowers the pH and reduces the zeta potential [44]. This phenomenon likely explains the concurrent decrease in pH and the zeta potential observed in samples stored at 40 °C.
Additionally, the formation of various degradation products during the auto-oxidation of polysorbate 80 in an aqueous medium may further contribute to the observed pH reduction [45]. The type of formulation and the preparation technique influence the drug quantity that can be incorporated into nanoparticles [46,47]. The entrapment efficiency could be considered a good result compared with the literature [48]. Hydrophobic drugs tend to concentrate in the inner core, and with the addition of oil into the core, entrapment efficiency tends to be higher [49].
The decreasing entrapment efficiency and drug content observed in the sample at 40 °C corroborates the pH decrease, as shown previously, since a pH decrease results in PCL hydrolysis, which leads to the inner release of the drug. Due to thymol’s volatility, it is possible to observe its degradation and a decrease in drug content at 40 °C.
The morphology of the nanocapsules can be attributed to the PCL, which forms a polymer coating around the inner components [50]. Also, microscopy shows that NPTMLs presented as a similar size to those determined by the dynamic light scattering technique.
Encapsulated drug release depends on its polymeric surface desorption, drug diffusion through the polymeric wall, and polymeric erosion or the combined diffusion and erosion process [51].
NPTMLs showed a slower profile release compared with the control. This can be explained by the fact that PCL is highly hydrophobic, which prevents drug diffusion to the medium [52]. Moreover, the caprylic triglycerides allow hydrophobic drugs to stay more concentrated in the core, minimizing drug release due to polymeric wall erosion [53].
The controlled drug release profile observed in NPTMLs is preferable since the formulation aims to remain on the skin for a long time, thereby reducing the need for reapplication. In this way, less reapplication leads to less exposure to excessive doses, which can cause adverse effects [54].
Porcine skin presents anatomical, genetic, and physiological similarities to human skin, which is why pigs are excellent animal models in some in vitro studies. The stratum corneum thickness of porcine skin varies from 30 to 140 μm, while the stratum corneum thickness of human skin ranges from 50 to 120 μm [55], making permeation study results in porcine and human skin very similar [56].
Thymol amounts from the control sample penetrated easily into skin layers. This is due to thymol’s high lipophilicity, which leads to a higher affinity for skin layers [57]. Higher NPTML penetration into the remaining skin was expected since smaller nanoparticles favor drug permeation through the skin [50].
The mass loss observed during the TGA analysis was due to sample volatilization and/or degradation during the heat process [58], corresponding to its complete evaporation [20]. HPβCD showed an initial mass loss related to cyclodextrin inner water evaporation and other mass loss between 300 °C and 380 °C, corresponding to its degradation [59]. Still, the expected mass loss in this sample was delayed, possibly due to the thymol encapsulation into cyclodextrin during the analysis by forming an in situ inclusion complex [60].
PCL degradation can explain the breakage of C-C bonding on the PCL structure. The NPTML TGA curve exhibited four stages of mass loss, which correspond to (1) water and non-encapsulated thymol, (2) thymol evaporation, (3) HPβCD and PCL degradation, and (4) residual component evaporation [61].
The residual water content after the sample lyophilization process can explain the anticipation of the first mass loss step [62]. Importantly, the second mass loss step at high temperatures indicates the stabilization of thymol, delaying drug evaporation [63].
The DSC analysis confirmed the drug’s entrapment in a polymeric matrix and revealed a possible interaction between the drug, cyclodextrin, and the polymeric matrix, when considering enthalpic changes. Pure thymol showed an enthalpy (ΔH) of 107.1 J/g [10].
The physical mixture’s thermal profile showed the expected thermal events of its components. The DSC curve, in turn, exhibited a thermal profile of the NPTMLs that was quite different from their physical mixture. The endotherm corresponding to the thymol phase transitions was markedly reduced to 2.33 J/g [64]. Such a result indicates a high level of interaction between thymol and the excipients used, which was intended for this formulation and corroborates previous results [65].
It is well known that synthetic and natural polymeric nanoparticles could control the drug release of numerous hydrophobic substances, directly affecting their efficacy. They could enhance molecule stability and load one or more active ingredients. Polymers can encapsulate, adsorb, disperse, or bind the active molecules, depending on the manufacturing process [21]. In this sense, thymol has been loaded into different nanoparticles, but different data have been obtained. In most cases, a controlled release was desired to improve its performance for different applications. For instance, thymol-loaded polymeric nanoparticles modified by oleic acid released approximately 65% of thymol in the first 12 h of the study [66]. Thymol was also loaded into solid lipid-polymer hybrid nanoparticles and incorporated into chitosan hydrogels, but 50% of thymol was released after 6 h of incubation [67]. PLA-loaded thymol nanoparticles were produced and 93% of the thymol was released in 6 h at 35 °C [68]. Even in lipid nanoparticles, 60% of the thymol was released in 18 h [69]. On the contrary, biogenic silica nanoparticles were produced with thymol, and 50% were released in 48 h [70]. Our study unequivocally demonstrates the ability of thymol-loaded polymeric nanocapsules to repel Rhipicephalus sanguineus nymphs by an impressive 60–70% for a sustained period of 2 h, firmly establishing their potential as a highly effective arthropod repellent. Our unwavering confidence in the safety of low-dose encapsulated thymol is underpinned by prior toxicity studies, which revealed no significant chronic toxicity concerns [71].
It is also essential to report that insect repellent containing natural products had a mean duration of repellency of less than 20 min against Aedes aegypti female mosquitoes [18]. Thus, further studies will be performed with mosquito species to evaluate the feasibility of using thymol-loaded nanoparticles as insect repellent.

5. Conclusions

The interfacial deposition of a preformed polymer technique allowed the development of thymol-loaded polymeric nanocapsules with repellent activity against R. sanguineus. The developed nanoformulation allowed high entrapment efficiency, homogenous size distribution, and a capacity to control thymol release and volatilization. Additionally, such a nanosystem proved to be stable at 4 °C for 90 days. Moreover, the formulation contains components that are of low toxicity to the skin, which is ideal for topical repellent application.

Author Contributions

A.M.R.S.: Writing—original draft. G.R.S.P.: Writing—original draft. L.C.N.L.: Writing—original draft. C.M.O.M.: Conceptualization, Writing—review & editing. B.N.M.: Writing—review & editing. S.F.T.: Conceptualization, Writing—review & editing. M.C.-F.: Conceptualization, Writing—review & editing. G.M.G.: Conceptualization, Writing—review & editing. T.G.: Conceptualization, Supervision, Funding, Writing—review & editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Project n. 402587/2021-9) and FAPDF (Fundação de Apoio à Pesquisa do Distrito Federal Induzida, Project n. 00193.000.00746/2021-08) for the financial support, and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for providing the scholarship for Amanda Malini.

Institutional Review Board Statement

This work was approved by the Ethics Committee on Animal Use of the UFG (CEUA/UFG) (Protocol n. 058/20).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data available upon request.

Acknowledgments

The authors acknowledge “Bonasa Alimentos” for providing porcine skin. The authors would also like to express their sincere gratitude to the Microscopy Laboratory at the University of Brasília (UnB) for the technical support and microscopy services provided during this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Coatings 14 01295 g001
Figure 1. NPTML stability. The samples were stored at 4 °C and 40 °C, and the parameters of (A) hydrodynamic diameter, (B) polydispersity index (PdI), (C) zeta potential, (D) pH, (E) encapsulation efficiency, and (F) drug content were evaluated for 90 days (n = 3).
Figure 1. NPTML stability. The samples were stored at 4 °C and 40 °C, and the parameters of (A) hydrodynamic diameter, (B) polydispersity index (PdI), (C) zeta potential, (D) pH, (E) encapsulation efficiency, and (F) drug content were evaluated for 90 days (n = 3).
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Figure 2. TEM images of thymol-loaded polymeric nanocapsules at different magnifications.
Figure 2. TEM images of thymol-loaded polymeric nanocapsules at different magnifications.
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Figure 3. Thymol release profile from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control over 24 h (n = 5).
Figure 3. Thymol release profile from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control over 24 h (n = 5).
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Figure 4. Thymol skin delivery from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control. After 2 h of skin treatment, thymol was recovered from the stratum corneum (SC), hair follicle (HF), and remaining skin (RS) (n = 5). ** p < 0.01.
Figure 4. Thymol skin delivery from thymol-loaded polymeric nanocapsules (NPTMLs) compared with the free-drug formulation used as a control. After 2 h of skin treatment, thymol was recovered from the stratum corneum (SC), hair follicle (HF), and remaining skin (RS) (n = 5). ** p < 0.01.
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Figure 5. First derivate of the TGA analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.
Figure 5. First derivate of the TGA analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.
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Figure 6. DSC analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.
Figure 6. DSC analysis of thymol-loaded polymeric nanocapsules (NPTMLs), the physical mixture, thymol, HPβCD, and polycaprolactone (PCL), as supplied.
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Table 1. Percentage (mean ± SD) of repellence against unfed R. sanguineus nymphs (n = 10) from ethanol, unloaded nanoparticles, thymol ethanolic solution, and thymol-loaded nanoparticles. * p < 0.05.
Table 1. Percentage (mean ± SD) of repellence against unfed R. sanguineus nymphs (n = 10) from ethanol, unloaded nanoparticles, thymol ethanolic solution, and thymol-loaded nanoparticles. * p < 0.05.
TimeNegative Control (Ethanol)Unloaded
Nanoparticles		Thymol Ethanolic SolutionThymol-Loaded Nanoparticles
1 min51.7 ± 16.636.7 ± 23.3 *66.7 ± 17.6 *68.3 ± 16.6 *
5 min47.9 ± 16.541.7 ± 16.266.7 ± 13.6 *73.3 ± 14.0 *
10 min45.8 ± 17.241.7 ± 21.168.3 ± 14.6 *71.7 ± 15.8 *
15 min50.0 ± 15.740.0 ± 16.160.0 ± 14.070.0 ± 20.5 *
1 h38.7 ± 14.435.4 ± 18.765.0 ± 19.9 *64.5 ± 13.9 *
2 h36.7 ± 18.933.3 ± 19.260.0 ± 21.1 *51.7 ± 19.9 *
24 h50.0 ± 19.250.0 ± 17.655.0 ± 19.345.0 ± 24.9
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Sales, A.M.R.; Pereira, G.R.S.; Lima, L.C.N.; Monteiro, C.M.O.; Matos, B.N.; Taveira, S.F.; Cunha-Filho, M.; Gelfuso, G.M.; Gratieri, T. Thymol-Loaded Polymeric Nanocapsules’ Repellent Activity on Nymphs of Rhipicephalus sanguineus Sensu Lato. Coatings 2024, 14, 1295. https://doi.org/10.3390/coatings14101295

AMA Style

Sales AMR, Pereira GRS, Lima LCN, Monteiro CMO, Matos BN, Taveira SF, Cunha-Filho M, Gelfuso GM, Gratieri T. Thymol-Loaded Polymeric Nanocapsules’ Repellent Activity on Nymphs of Rhipicephalus sanguineus Sensu Lato. Coatings. 2024; 14(10):1295. https://doi.org/10.3390/coatings14101295

Chicago/Turabian Style

Sales, Amanda M. R., Gessyka R. S. Pereira, Lais C. N. Lima, Caio M. O. Monteiro, Breno N. Matos, Stephânia F. Taveira, Marcilio Cunha-Filho, Guilherme M. Gelfuso, and Tais Gratieri. 2024. "Thymol-Loaded Polymeric Nanocapsules’ Repellent Activity on Nymphs of Rhipicephalus sanguineus Sensu Lato" Coatings 14, no. 10: 1295. https://doi.org/10.3390/coatings14101295

APA Style

Sales, A. M. R., Pereira, G. R. S., Lima, L. C. N., Monteiro, C. M. O., Matos, B. N., Taveira, S. F., Cunha-Filho, M., Gelfuso, G. M., & Gratieri, T. (2024). Thymol-Loaded Polymeric Nanocapsules’ Repellent Activity on Nymphs of Rhipicephalus sanguineus Sensu Lato. Coatings, 14(10), 1295. https://doi.org/10.3390/coatings14101295

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