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Hydrophobic nanoprecipitates formed by benzoylphenylureas and β-cyclodextrin inclusion compounds: synthesis, characterization and toxicity against aedes aegypti larvae

The aim of this work was to synthesize and characterize the inclusion compounds formed by the complexation of β-cyclodextrin (βCD) with insecticides from the class of benzoylphenylureas (BPUs), named novaluron (NOV) and diflubenzuron (DIF), beyond evaluate their larvicidal activity against Aedes aegypti larvae. Solid state characterization by FTIR showed changes in the main peaks of BPUs and βCD, suggesting the formation of inclusion compounds in solid phase. DTA and TGA thermal analysis showed changes in temperatures of BPUs decomposition as result of molecular interactions. 1H NMR experiments allowed to observe the occurrence of interactions in solution through changes in chemical shifts of BPUs aromatic hydrogens. However, the presence of H–H intermolecular correlations in 2D ROESY was found only for the DIF/βCD complex, suggesting different topology for each complex. Such hypothesis was corroborated by thermodynamic analysis using ITC, which showed different profile of titration curves, beyond endothermic and exothermic interactions for NOV/βCD and DIF/βCD complexes, respectively. DLS titrations of BPUs or BPUs/βCD DMSO solutions in aqueous solution demonstrated that the spontaneously formed hydrophobic nanoprecipitates (HNPs) have different profile of sizes depending on the BPU/βCD system, corroborating also with the hypothesis about the existence of different topologies for each complex. Finally, the HNPs of inclusion compounds showed to be more efficient than free BPUs, allowing proposing a new insecticide formulation.


Introduction
Benzoylphenylureas (BPUs) is an important class of low molecular weight insecticides, which are used to combat several arthropods, such as mosquitoes, cockroaches, fleas, ants and termites in wide world, including the Aedes aegypti, the vector of diseases as yellow fever, dengue, chikungunya and zika [1,2,3,4].
These compounds belong to the group of insect growth regulators (IGRs), whose the mechanism of action is based on the inhibition of chitin synthesis during the insect larval stages, causing feeding difficulty, malformation (of pupae and adults), and complete or partial inhibition of molt [5]. Moreover, they are more selective than classical insecticides once they act on structures that do not exist in vertebrates, reducing thereby their toxicity to mammals [1]. The absorption of these compounds by the insect occurs mainly through ingestion. However, absorption cases by direct contact with the integument have been reported [6].
Some studies have demonstrated that IGRs are effective even in the control of vectors with a recognized resistance to conventional insecticides, due to their differential action mechanism. For this reason, BPUs have been widely used in Brazilian regions where the resistance to the organophosphate insecticides was detected [9,10].
Recently, Benilato (2015) showed that upon the treating of successive Aedes aegypti generations with a predetermined dosages of diflubenzuron, it was identified a resistance development to this compound [11]. This result justifies the need for constant evaluation of the insecticides advocated by the WHO, as well as the need of continuous search for improvement of existing insecticides.
From the perspective of development of new formulations, the strategy of preparing controlled release systems for environmental purposes, via host/guest interactions, have been proposed [12]. In this context, complexation of insecticides with cyclodextrins (CDs) could be an important strategy to improvement of current formulations [13,14,15]. CDs ( Fig. 2) are cyclic oligosaccharides composed usually of six, seven or eight units of glucopyranoses linked by α-1,4-type glycosidic bonds, being called of α-cyclodextrins (αCD), β-cyclodextrin (βCD) and γ-cyclodextrin (γCD), respectively (Fig. 2). As result of their conformations, such compounds acquire the truncated cone shape, with a central cavity that allows to accommodate hydrophobic portions of the guest molecules. Among the natural CDs, βCD is the most used in formulations due to the diameter of its cavity (%6.5 Å) and its best cost-benefit ratio when compared to other commercially available ones [16]. The formation of the complex between βCD and the guest molecule (inclusion compound) can cause changes in the physicochemical properties of the guest molecules (specially solubility, stability and reactivity), affecting their responses in biological systems [17,18,19].
Thus, aiming the developing of new formulations to overcome the problems about A. aegypti resistance against conventional insecticides, in this work we have synthetized and characterized the inclusion compounds formed between βCD and the benzoylphenylureas DIF and NOV, aiming to improve their activity against Aedes aegypti larvaes. Concerning the low aqueous solubility of these BPUs, even in presence of βCD, additionally we have invoked the simple concept of hydrophobic nanoprecipitates (HNP) [13,20,21] by using an organic co-solvent (DMSO) to promote the initial solubilization of compounds and subsequent precipitation of nanostructured material by mixture with water.
Initially, the inclusion compounds were synthetized by coprecipitation method followed by lyophilization. The stability of the compounds were investigated by thermogravimetric and differential thermal analysis (TGA and DTA) while the topology of the complex in solid state and in solution were evaluated by FTIR and NMR ( 1 H and 2D-ROESY) respectively. The stoichiometry of the complexes and thermodynamic parameters of complexation were determined by Isothermal titration calorimetry (ITC). The HNPs, prepared by mixture of DMSO solution of BPUs or their respective inclusion compounds in water were characterized by DLS measurements. Finally, the larvicidal activity of HNPs formed by BPUs or their respective inclusion compounds were evaluated against 4 th instar larvae and emergence inhibition of Aedes aegypti mosquitoes.

Materials
Novaluron (purity 95.20%) and diflubenzuron (purity 96.00%) were respectively extracted and purified from the commercial formulations (Mosquilon®-Bayer) and (Dimilin®-Chemtura). The purity of these products was determined by HPLC comparing with novaluron and diflubenzuron standards purchased from Sigma-Aldrich ® . The βCD was purchased from Sigma-Aldrich ® (St. Louis, MO, EUA). All other chemical reagents were purchased from reputable companies (Vetec, Synth and J.T. Baker) and used as received. During all experiments, Milli-Q ® water was used.

Preparation of the inclusion compounds
The inclusion compounds between BPUs and βCD were prepared by co-precipitation method followed by liophilization. Briefly, equimolar amounts of the host molecule (DIF or NOV) and βCD were separately solubilized in ethanol and Milli-Q water, respectively. This proportion was chosen after the determination of stoichiometry by ITC experiment, as showed below in section 2.4.2.
The ethanolic solution of the host molecule was poured into the aqueous βCD solution and submitted to stirring during 24 h. Finally, the suspension formed was placed in rotary evaporator to remove the ethanol, and then submitted to the freeze-drying process to obtain the desired inclusion compound (NOV/βCD or DIF/βCD).

Preparation of the hydrophobic nanoprecipitates
HNPs were prepared by dissolution of appropriate amounts of NOV, DIF, NOV/βCD or DIF/βCD in DMSO (as cossolvent) to promote the initial solubilization of compounds. Further, these solutions were mixed in water and subsequent precipitation of nanostructured material were spontaneously observed. These compounds were used in bioassays against A. aegypti.

Solid-state characterization
The FTIR spectra of DIF, NOV, βCD, NOV/βCD, DIF/βCD, and physical mixtures (PM) at molar ratio of 1:1 were obtained using a Perkin Elmer Fourier transform spectrometer (Spectrum Two™ model). The spectra in KBr discs were recorded in range from 4000 to 400 cm À1 with a resolution of 2 cm À1 as the mean of 16 consecutive scans. The Perkin Elmer Spectrum ES program (application version: 10.03.08.0133) was used for the spectra acquisition and processing.
Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) for DIF, NOV, βCD, DIF/βCD, NOV/βCD and physical mixtures (PM) at molar ratio of 1:1 were performed using a simultaneous TGA/ DTA HITACHI-STA 7200 RV thermoanalytical module. The analytical conditions were: air atmosphere at 300 mL/min, with a temperature ramp of 35 C-800 C, a heating rate of 10 C/min, and mass of %3.0 mg of the samples in a platinum crucible. As reference, the empty platinum crucible was used. The data were exported to Microcal Origin 9.0 for editing.

Isothermal Titration Calorimetry (ITC)
The thermodynamic parameters involved in the inclusion compounds formation between guest molecules and βCD in solution were determined using isothermal calorimetric titration at 25 C. injection of 1.0 μL was discarded in order to eliminate the diffusion material effects from the syringe to the cell and vice-versa. After the experiments, the titration curves were subtracted from the respective blanks in order to mathematically eliminate the interaction effects of the guest molecules with the solvent. The data were treated using Microcal Origin 7.0 program for ITC.

Nuclear magnetic resonance (NMR)
NMR spectra were recorded at 27.0 C on a Bruker DRX 400 -AVANCE spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at 400 MHz, equipped with a 5 mm inverse probe with a z-gradient coil. 1 H NMR spectra were achieved using the WATERGATE technique for suppression of the residual water signal. The nuclear Overhauser enhancement spectroscopy (2D NOESY) experiments (mixing time 500 ms) were acquired using standard experiments from the spectrometer library. All NMR experiments were performed in the Laborat orio de Ressonância Magn etica Nuclear de Alta Resolução -LAREMAR -DQ/ ICEx-UFMG.

Size determination of HNPs by dynamic light scattering (DLS)
DLS experiments were performed in a Malvern Zetasizer Nano ZS particle analyzer, using polyethylene square cells to measure the average hydrodynamic diameter (D h ) of NOV, DIF, NOV/βCD and DIF/βCD hydrophobic nanoprecipitates spontaneously formed by the mixture of their DMSO solutions in water. Initially, it was prepared solutions containing 1.0 mg of NOV, DIF or equimolar amounts of NOV/βCD or DIF/βCD in 0.5 mL of DMSO. Then, 25 injections of 20.0 μL of these solutions were titrated in 2.0 mL of Milli-Q water, with subsequent measurements of the HNPs hydrodynamic diameter by DLS. These samples were submitted to a monochromatic light (4 mW He-Ne laser, wavelength 633 nm) and the scattered light intensity was measured at an angle of 90 . The D h were determined by the average of five independent measurements, each of them obtained as the mean of 10 counts, with intervals of 2 s each.

Bioassays involving larvae of the Aedes aegypti mosquito
The Aedes aegypti (PPCampos strains) eggs were donated by the Department of General Biology from Federal University of Viçosa, MG -Brazil. They were placed in plastic basins containing 2L of dechlorinated water, with a temperature of 26 AE 2 C, RH> 70% and photoperiod of 12L:12D. After 24 h, the 1 st instar larvae (L1) were transferred to another basin with 2 L of dechlorinated water and approximately 118 mg of food (Alcon GoldFish Colour Bits). Thereafter, the water was changed daily by replenishing the food until individuals reached the stage of development required for each test, 3 rd instar larvae (L3) for Emergency inhibition assay or 4 th instar larvae (L4).
The larvicidal activity of the obtained HNPs described in section 2.3, on 4 th instar larvae, was evaluated using the method recommended by the World Health Organization with small modifications [22]. Briefly, twenty 4 th instar larvae were added to a beaker (250 mL) together with 50 mL of dechlorinated water and food. Thereon, 50 mL of each sample stock solution was added to the beaker so that suspension reached the desired concentration of the compound and 1% DMSO in volume. The nominal concentrations used in experiments for each HNP were 10 2 , 10 1 , 10 0 , 10 À1 , 10 À2 and 10 À3 μmol/L. Mortality of the larvae was determined after 24 h and 48 h of incubation at 26 AE 2 C and RH > 70%. The larvae were considered dead when they did not demonstrate any response to the external stimulus. Three replicates were performed in three independent experiments. The mortality percentage curves versus the compound concentration logarithm were constructed using the GraphPad Prism 5.0 software. The lethal concentration value (LC 50 ) at 24 h and 48 h was calculated by Probit algorithm using the same software.
The emergency inhibition of Aedes aegypti mosquitoes in presence of HNPs of DIF, NOV, βCD, DIF/βCD and NOV/βCD was evaluated also using a method recommended by the World Health Organization with small modifications [22]. In this experiment, twenty 3 rd instar larvae were added to a beaker (250 mL) together with 50 mL of dechlorinated water and food. Then, 50 mL of each sample stock solution was added to the beaker in order to obtain the desired concentration of the compound (10 0 , 10 À1 , 10 À2 , 10 À3 , 10 À4 and 10 À5 μmol/L) and 1% of DMSO in volume. The mortality of the insects was verified at intervals of every 12 hours until the emergence of all adults in the control (nearly 10 days after the experiment beginning) [22].
During the experiment, the information about the development stage of insects were also collected. Three replicates were performed in three respectively, were observed. These data are similar to those previously described in literature [23]. Due to the molecular similarities between NOV and DIF, both compounds showed FTIR bands or peaks at the same range: 3230 and 3120 cm À1 , attributed to the stretching of N-H bonds; 1706 and 1692 cm À1 , attributed to the stretching of C¼O bonds; 1600-1400 cm À1 , attributed to vibrations of aromatic rings; and at 800-600 cm À1 , attributed to characteristics of the C-Cl bonds. The main difference between the two  spectra is in the range of 1400-1000 cm À1 , attributed to vibrations of CF 2 and CF 3 groups present only in the NOV molecule.
In the physical mixture's spectra, no change in the positions of the main bands was observed in relation to free benzoylphenylureas or free βCD. Moreover, the observed profile can be ascribed as the overlapping of bands of BPUs and βCD.
In the FTIR spectra of the inclusion compounds, significant changes in the shape and position of the vibrational bands were observed which in turn did not superimpose with the FTIR spectra of free species or their PMs. For the NOV/βCD and DIF/βCD complexes, the OH bands of βCD at 3386 cm À1 were sharper if compared with the free βCD, as result of βCD-βCD hydrogen bonds breakdown in solid state, after formation of new interactions. Moreover, it was observed disappearance of ν(C-H) and δ(C-H) bands of βCD, at 1650 and 860 cm À1 , confirming the existence of new vibrational profile for the cyclodextrins after interactions.
Angular deformations disappearance of aromatic C-F, C-H and C-Cl bonds of NOV, at around 1020, 990 and 680 cm À1 respectively suggest interactions of aromatic groups with the cavity of βCD. Other NOV or DIF bands, at 796, 778, 766, 722, 595, 539 and 500 cm À1 , had reduced or missed their intensities (Fig. 3). For the DIF/βCD system, it was observed reduction or disappearance of ν(C-N) peaks at 1138 and 1096 cm À1 , ν(C-F) at 1015 cm À1 , δ(C-H) at 830 and ν(C-Cl) at 593 cm À1 (Fig. 4). Transitions observed for free βCD are consistent with those previously described in literature [13,24].
In TGA curves of NOV and DIF, a single loss of mass event was observed in the range from 170 C to 245 C, which has been attributed to the one step decomposition process. The two curves similarity was result from the structural similarity. On the other hand, DTA curves showed some differences. For NOV, it was possible observe two endothermic peaks, being the first one attributed to its fusion, while the second one, attributed to its decomposition. For DIF, only a broad endothermic event, ascribed as the simultaneous fusion and decomposition of compound, has been observed.
In the TGA and DTA curves of PMs, there are four events related to βCD and benzoylphenylureas, with low differences if compared with free compounds. However, the results for NOV/βCD and DIF/βCD inclusion compounds showed greater differences in thermal profiles. The maximum of decomposition peak was reduced from 237 C to 224 C and from 244 C to 235 C for NOV and DIF respectively. Moreover, the fusion decomposition peaks for both insecticides, as well as the decomposition event of βCD, were all attenuated in the complexes, suggesting the amorphization of solid phase resulting from the formation of new interactions.

Isothermal Titration Calorimetry
Changes in thermodynamic properties due to interactions between compounds can be properly measured by Isothermal Titration Calorimetry (ITC) [25], which allows to determinate the thermodynamic parameters of binding, as free energy of Gibbs (Δ b G o ), enthalpy (Δ b H o ) and entropy (TΔ b S o ). Besides, the binding constant (K b ) and the reaction stoichiometry (N) can be calculated using the Wiseman isotherm to model [26].
The titration curves of NOV and DIF, at 30.0 mM, into the ITC cell charged with βCD solution at 2.0 mM are shown in Figures 7A-B and 8A-B. βCD was chosen as titrand, since high concentrations of cyclodextrins in presence of low amounts of guest molecules can induce selfaggregation [27,28,29,30,31].
As can be seen in Figs. 7A and 8A, the heats of titration (injection of NOV or DIF solutions in βCD solution), were very different from the heats of dilution (injection of NOV or DIF solutions in solvent). These differences are indicative of interactions in solution, so that the heat measured in ITC experiments are resultant from establishment of new intermolecular interactions. After subtraction of blank experiments, Figs. 7B and 8B were obtained and used to calculate the stoichiometry and thermodynamic parameters by non-linear fitting with Wiseman Isotherm.
In both systems, the stoichiometric coefficient was close to unity (N NOV/βCD ¼ 0.92 AE 0.04 and N DIF/βCD ¼ 1.01 AE 0.02), indicating that the 1:1 stoichiometries are the most favored in the used solvent. Thus, it is justified the preparation of inclusion compounds with this molar ratio for biological experiments, using DMSO. In overall, the equilibrium constants determined for the systems were in the same magnitude as that found in literature for other inclusion compounds [21,32,33].
However, the equilibrium constants were very different for the two binding process, suggesting the occurrence of different molecular topology, in spite of the molecular similarity (K NOV/βCD ¼ 510.0 AE 31.8 and K DIF/βCD ¼ 65. In both cases the entropy contribution suggests desolvation of precursors and releasing of solvent molecules upon inclusion. The solvent molecules must gain rotational and translational degrees of freedom, causing an increase in the entropy (disorder) of the system [34]. However, the explanation for the enthalpic differences relies on the fact that the 1,1,2-trifluoro-2-trifluoro-methoxyethoxy group makes NOV much more hydrophobic molecule than DIF (DIF is 27 times more soluble than NOV in water), increasing therefore, its affinity by the βCD. The presence of this fluorocarbon group explains the endothermicity of the NOV/βCD interaction, once is necessary an energy consumption for the partial desolvation of these groups during inclusion.

Structural characterization by NMR spectroscopy
It is well known in the literature that the formation of supramolecular complexes with cyclodextrins generates changes on the electron density of the hydrogen nuclei involved in the interactions, which is reflected in the NMR spectra as changes of chemical displacement or extension of the peaks. In Figs. 9 and 10 in supplementary material are shown the 1 H NMR spectra of NOV, DIF, βCD, NOV/βCD and DIF/βCD. The 1 H NMR spectra of NOV and DIF were similar to that provided by Sigma-Aldrich ® while the βCD spectrum was similar to others found in literature [21]. NOV/βCD complex showed changes in chemical shifts of OH(3), OH(6) and CH(1) hydrogen atoms of βCD, as well as of NH(9), CH (15) and CH(18) hydrogen atoms of NOV. In DIF/βCD complex it was observed changes in the chemical shifts of CH(1), CH(5), OH(2), OH(3) and OH(6) of the βCD and in NH(10), CH(2) and CH(6) hydrogen atoms of DIF. In addition, OH(2) hydrogen atom have been found to have better defined coupling.
The ROESY experiments were performed in order to identify the presence of dipolar correlation until 5Å between the hydrogen atoms and therefore confirm the interaction between the host and guest. Fig. 11 shows ROESY correlations between the OH(6) hydrogen atom of the βCD and the hydrogen atoms bound to the NH(8) hydrogen atom of the DIF, as well as correlations among the OH(2) and OH(3) of the βCD and NH (8) and NH(10) hydrogen atoms of DIF. Nevertheless, NOV/βCD system did not show any correlation in 2D spectrum (data not shown), in spite of the use of the same parameters in the two ROESY experiments. This suggests that the mode of DIF interaction with the βCD must be different from that observed for NOV/βCD system as proposed in Fig. 12 in Supplementary material.
A possible explanation for these differences is the inclusion mode of NOV by the fluorocarbon group, which is invisible to the 1 H NMR. This hypothesis explains the great difference in thermodynamic parameters as described in ITC experiments.

Characterization of hydrophobic nanoprecipitates
Considering that the insecticides described in the present work, even in presence of βCD, presented very low solubilities, it was proposed the strategy to use them in form of hydrophobic nanoprecipitates. Such kind of materials are formed when a low water soluble substance, previously dissolved in an organic solvent (such as DMSO), is added in water at low co-solvent concentration and volume [13,21]. They can be stabilized in an aqueous environment by the appropriate concentration control of the solid phase and mixture ratio of solvent/cosolvent. Thus, in order to evaluate the ability of βCD modulate the self-aggregation of benzoylphenylureas in water/DMSO mixture, the size of the so formed aggregates was investigated by measurements of average hydrodynamic diameter (D h ). Fig. 13 shows the DLS titrations of NOV, DIF, NOV/βCD and DIF/βCD DMSO solutions in ultrapure (Milli-Q ® ) water. It was observed in all systems that the particle size increased with concentration of titrant. This behavior may be associated with the gradual increase of collision probability between the particles, with subsequent coalescence.
Titration of NOV and NOV/βCD showed that at concentrations lower than 600 μmol/L, the inclusion compound presents lower values of D h , suggesting higher colloidal stability for this nanocoprecipitate. As consequence from the size reduction, a larger surface area of the particle is exposed allowing a new pattern of interactions with the medium. These smaller particle size can be attributed to the presence of βCD molecules on the particles surface, favoring greater interactions with the solvent and reducing the particle-particle interaction. For the DIF, it was observed that the D h values were higher in presence of βCD, but with very lower polidispersity if compared with DIF/ βCD titration. These data show once again that DIF must has a different profile of interaction with βCD if compared with NOV/βCD system. This difference was attributed to the absence of the 1,1,2-trifluoro-2-trifluoromethoxy-ethoxy group in the DIF molecule, which should generate a novel host-guest interaction mode, producing colloidal changes in the hydrophobic nanoprecipitates.
Based on these data and on the knowledge about the fast and indiscriminate feeding of larvaes in initial stages of their life, the presence of hydrophobic nanoprecipitates could be a way to attract them, actin as bait for larvaes.

Larvicidal assay in 4 th instar larvae
According with recommendation from the WHO, the 4 th instar larvae bioassay is the standard test for evaluate the larvicidal activity of new compounds or insecticide formulations, before their approbation to use in the environment. Thus, the HNPs of inclusion compounds, prepared in DMSO/water solvent were evaluated and compared with the HNPs of their BPUs precursors. In Fig. 14 are shown the curves of % mortality as a function of log concentration of the tested compound (NOV, NOV/βCD, DIF or DIF/βCD).
The obtained dose-response curves demonstrate that both free compounds and DIF/βCD have poor larvicidal activity in this bioassay. As mentioned above, the acute effect is not expected for compounds of this class, so that their mechanism of action are based on the inhibition of chitin synthesis during the insect larval stages. This can be confirmed by    This suggests that for NOV, the strategy of preparing the HNP of inclusion compound might have a new acute effect on dose reduction. However, new studies would be necessary to confirm this hypothesis.

Evaluation of the emergency inhibition
Compounds whose mechanism of action is related to changes in the process of growth and development of insects, such as BPUs, will require monitoring the development of the insect to verify its action. For this reason, the WHO advocated the implementation of the emergency inhibition test for compounds in which the mechanism of action is the regulation of insect growth [22]. Fig. 15 show the cumulative mortality curves as a function of exposure time during emergency inhibition bioassay for NOV, DIF, NOV/βCD and DIF/βCD HNPs. The negative controls used were dechlorinated water, 1% aqueous DMSO solution and aqueous βCD solution at the highest concentration used in the tests.
For all the compounds, no significant mortality was observed for larvae in the first 12 hours of experiment. However, after the ecdysis period, it was observed a significant mortality, mainly, at concentrations of 10 nmol/L for both pure and the complexated compounds, which matches with the action mechanism of compounds by growth regulation.
As can be seen in Fig. 15A, the NOV/βCD HNP is able to cause a delay in ecdysis if compared with free NOV, representing a new advantage. Moreover, Fig. 15B show a lower error bar of the points in mortality curve for the DIF/βCD HNP, indicating a more robust mortality profile in βCD presence.
In Table 1 are shown the necessary concentration to inhibit the emergence of 50% of the Aedes aegypti mosquito's insect population (% IE 50 ) for NOV, DIF, NOV/βCD and DIF/βCD HNPs.
The %IE 50 values observed for the compounds were all in ppb magnitude. The values obtained for NOV and DIF are close to those presented by other works related in the literature, 0.135 ppb and 1.59 ppb [35], respectively. The data in Table 1 show that the inclusion compounds HNPs were more potent than free molecules. This demonstrates that the strategy of preparing HNP inclusion compound with BPUs was advantageous, leading to an increase of toxicity against Aedes aegypti larvae.

Conclusions
In the present work, two strategies were employed in order to develop new insecticide formulations based on benzoylphenylureas. The first one refers to complexation of BPUs with βCD in order to obtain their respective inclusion compounds, which were properly characterized. The second one was based on the production of hydrophobic nanoprecipitates through dispersion of DMSO solutions of these compounds in water. The results obtained during the characterizations confirmed the success in the preparation of the compounds, in spite of the differences in modes of inclusion for the two compounds, as showed by NMR and ITC experiments. These differences were attributed to the presence of 1,1,2-trifluoro-2-trifluoro-methoxyethoxy moiety in the NOV, which is responsible for reducing its solubility 27 times if compared with DIF, as described in literature. It is believed that this functional group is also responsible to give rise the drastic difference in the hydrophobic nanoprecipitates of the inclusion compounds, as demonstrated by DLS titrations. Results of larvicidal tests, both at the L4 stage and the emergency inhibition, showed that the inclusion compounds were more potent than their precursors, bringing other advantages as delaying in the ecdyses for NOV/βCD and becoming the mortality profile much more robust for DIF/ βCD complex. The present results show that the preparation of new formulations from already used insecticides led to an increase in larvicidal potency after encapsulation, possibly producing an acute effect for NOV/ βCD system. These results are, at the best, tentative. Deeper research must be performed in order to bring out more conclusive results.

Author contribution statement
Angelo M arcio Leite Denadai: Conceived and designed the experiments; Wrote the paper.