Avermectins

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Agricultural and Environmental Chemistry

Hydrophobic nanoprecipitates of #-cyclodextrin/avermectins inclusion compounds reveal insecticide activity against Aedes aegypti larvae and low toxicity against fibroblasts Ana Maria dos Santos Moreira, Vanessa Bittencourt, Fábio Costa, Maria Elena de Lima, Miriam Lopes, Warley S. Borges, Gustavo Martins, Jeferson da Silva, Ângelo M. Leite Denadai, and Keyller Bastos Borges J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01300 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Journal of Agricultural and Food Chemistry

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Hydrophobic nanoprecipitates of β-cyclodextrin/avermectins inclusion

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compounds reveal insecticide activity against Aedes aegypti larvae and low

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toxicity against fibroblasts

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Ana M. dos Santos Moreira †,§ Vanessa C. E. Bittencourt§, Fábio L. S. Costa‡, Maria Elena de

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Lima‡, Miriam T. P. Lopes‡, Warley S. Borges∥, Gustavo F. Martins#, Clébio S. Nascimento

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Jr†. Jeferson G. da Silva§, Ângelo M. L. Denadai*§, and Keyller B. Borges*†

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†

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Dom Bosco, 36301-160, São João del-Rei, MG, Brazil.

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§

Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, Campus

Departamento de Farmácia, Universidade Federal de Juiz de Fora, Campus Governador

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Valadares, 35010-177, Governador Valadares, MG, Brazil.

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‡

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901, Belo Horizonte, MG, Brazil.

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∥

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29075-910, Vitória, ES, Brazil.

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#

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36570-900, Viçosa, MG, Brazil.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, 31270-

Departamento de Química, Universidade Federal do Espírito Santo, Campus de Goiabeiras,

Departamento de Biologia Geral, Universidade Federal de Viçosa, Campus Universitário,

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*Corresponding author:

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Prof. Keyller Bastos Borges, PhD, Departamento de Ciências Naturais, Universidade Federal

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de São João del-Rei, Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160, São

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João del-Rei, Minas Gerais, Brazil. *e-mail: [email protected]. Phone: +55 32 3379 – 2481

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/ Fax: +55 32 3379 – 2483.

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Prof. Ângelo M L Denadai, PhD, Departamento de Farmácia. Universidade Federal de Juiz de

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Fora – Campus Governador Valadares, MG, Brazil. e-mail: [email protected].

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Phone: +55-(33)33406501, Fax: +55-(32)2102-3802.

1 ACS Paragon Plus Environment


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ABSTRACT: In the present work, hydrophobic nanoprecipitates (HNPs) of inclusion

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complexes formed between β-cyclodextrin (βCD) and the avermectins (AVMs) named

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eprinomectin (EPRI) and ivermectin (IVER) were synthesized and characterized, and their

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larvicidal activity against Aedes aegypti and human safety against fibroblasts were evaluated.

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Initially, thermogravimetric analysis/differential thermal analysis data revealed that inclusion

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increased the thermal stability of AVMs in the presence of βCD. Nuclear magnetic resonance

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experiments and density functional theory calculations pointed out the inclusion of the

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benzofuran ring of the two AVMs in the βCD cavity. Isothermal titration calorimetry

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experiments allowed identification of different binding constants for EPRI/βCD (Kb = 1060)

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and βCD/IVER (Kb = 1700) systems, despite the structural similarity. Dynamic light

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scattering titrations of AVMs’ dimethyl sulfoxide solution in βCD aqueous solution

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demonstrated that the formed HNPs have lower sizes in the presence of βCD. Finally, the

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inclusion of EPRI in βCD increased its larval toxicity and reduced its human cytotoxicity

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while for IVER/βCD no beneficial effect was observed upon inclusion. These results were

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rationalized in terms of structural differences between the two molecules. Finally, the

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EPRI/βCD complex has great potential as an insecticide against Aedes aegypti larvae with

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high human safety.

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KEYWORDS: avermectins, cyclodextrins, inclusion compounds, larvicidal activity, human

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safety.

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INTRODUCTION

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Avermectins (AVMs, Figure S1) are 16-membered macrocyclic lactones, obtained by

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fermentation from Streptomyces avermitilis, which have potent anthelmintic and insecticide

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activities. They have been widely used as excellent parasiticides in veterinary medicine and as

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pesticides in agriculture and horticulture.1,2 The main mechanism by which the AVMs exert

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their toxic effect in insects is by binding ligand-gated chloride ion channels and binding of

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glutamate-gated chloride ion channels that are specific to invertebrates, causing an influx of

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chloride ions into the parasite neurons and leading thereby to hyperpolarization, paralysis, and

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death. This explains its relatively low toxicity to humans.3,4

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However, low concentrations of AVMs in the environment can cause several biological

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effects and significantly affect nontarget organisms in the soil and aqueous systems, including

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sediments.5,6 Natural AVM, commonly called abamectin, is a broad-spectrum pesticide,

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which has been widely used in agricultural fields and has hazardous impacts, especially on

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human beings.1–3 Ivermectin (IVER, Figure S1) and eprinomectin (EPRI, Figure S1) are

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semisynthetic derivatives of abamectin, obtained by hydrogenation of the double bond in the

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C22–C23 position, or substitution of OH group in the “C4“ position by the acetylamino group,

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respectively.7

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IVER was the first to be introduced commercially for the control of endoparasitic

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nematodes and ectoparasitic arthropods in livestock.8 It also shows excellent efficacy and

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high tolerability in the treatment of parasite infestations in human diseases.3,9 EPRI was

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developed exclusively for veterinary medical use, being a potent parasiticide, especially in

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bovines, in which it presents low elimination in cows’ milk.10,11 Furthermore, AVMs have

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demonstrated larvicide and adulticide activities for several arthropod species, with sublethal

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effects, including malformation, limited mobility, reduction in fertility, decrease in

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oviposition, reduction in blood meal frequency, and sensory and motor disorders.12,13



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Among the various arthropods that cause risks to health public, Aedes aegypti has occupied

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a prominent position. This mosquito is one of the main vectors of numerous arboviruses to

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humans, including dengue, urban yellow fever, chikungunya fever, and more recently zika.14

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Considering that no vaccine is properly available against chikungunya and zika viruses, the

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main way to combat these diseases is through control of the insect vector. For this purpose,

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chemical insecticides of diverse classes, such as organophosphorates, carbamates, and

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benzoylphenylureas have been usually employed for the elimination of the larval stage,

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especially due to its high vulnerability and minor risk of human contact.15 However, the

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indiscriminate use of these compounds has resulted in the increase of selection pressure on the

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mosquitoes,16 leading to the development of insecticide resistance.17,18

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This fact has encouraged the evaluation of the larvicidal activity of insecticides that are

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already being used for other types of arthropods, as well as the improvement of the existing

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formulations. In this scenario, the strategy of developing inclusion compounds between

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AVMs and cyclodextrins (CDs) could improve the bioavailability of these compounds, to be a

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possible strategy to overcome the problem of the pressure selection.

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CDs are cyclic oligosaccharides consisting of six to eight glucopyranose units linked by an

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α-1,4-glycosidic bond.19,20 The most common CDs are α, β, and γCDs, which are composed

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of six, seven, and eight glucopyranose units, respectively. These compounds display a toroidal

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form with a polar outer surface and a nonpolar interior cavity. The size of the CDs’ cavity

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allows the complexation of guest molecules or moieties, and, therefore, they can form

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inclusion complexes stabilized by noncovalent interactions with a wide variety of compounds.

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The inclusion of insecticides in βCD has been successfully applied in several works.21,22

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Among the main benefits reported by the authors are the increase in solubility and stability,

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controlled release profile, protection against photodegradation, and reduction of human

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toxicity, not affecting directly the drug’s mechanism of action. Thus, it is expected that the


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binding of guest molecule with biological site occur in free form, after dissociation of the

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inclusion complex at vicinity of site of interaction.

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In the case of very low water-solubility compounds such as the AVMs, it is also hoped that

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the complexation with CDs might reduce the size of the hydrophobic nanoprecipitates (HNPs)

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spontaneously obtained in organic/water mixture of solvents.

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Based on the above information, in the present work, we report the syntheses,

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characterization, and biological activities of HNPs of inclusion compounds formed between

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βCD and IVER and EPRI. Initially, the inclusion compounds were obtained by a

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coprecipitation method combined with lyophilization. The structure and stability of the

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compounds were investigated by Fourier transform infrared (FTIR) spectroscopy with

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attenuated total reflection (ATR), nuclear magnetic resonance (NMR) (1H and 2D-ROESY),

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thermogravimetric and differential thermal analysis (TGA and DTA, respectively). Isothermal

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titration calorimetry (ITC) and semiempirical and density functional theory (DFT)

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calculations were employed to determine the most stable stoichiometry and energies of

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interaction. The size and zeta potential of HNPs in dimethyl sulfoxide (DMSO)/water solution

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were characterized by dynamic light scattering (DLS) and zeta potential (ZP) titrations.

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Finally, bioassays were undertaken to evaluate the insecticide activity against first and fourth

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instar larvae of Aedes aegypti mosquitoes. In addition, the toxicological safety of the

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compounds was evaluated against fibroblasts using 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl)-

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2H-tetrazolium (MTT)-reduction assays.

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EXPERIMENTAL SECTION

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Materials. EPRI (Pestanal®, analytical standard) assay HPLC 97.7 area% (97.34% w/w,

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B1a + B1b) and IVER (Pharmaceutical Secondary Standard, Certified Reference Material)

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98.97% B1a were obtained from Sigma-Aldrich® (Steinheim, Germany): and used without



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any treatment. βCD was purchased from Sigma-Aldrich® (St. Louis, MO, USA). All other

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chemical reagents were of analytical grade and used as received. During all the experiments,

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ultrapure water was used (Milli-Q® Plus system, Bedford, MA, USA).

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Preparation of the inclusion complexes. The inclusion complexes formed between

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AVMs with βCD (IVER/βCD and EPRI/βCD) were prepared by a coprecipitation method.

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Briefly, IVER or EPRI was dissolved in ethanol and βCD was dissolved in ultrapure water at

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1:1 mole ratio. The ethanol solution of AVMs was poured into the aqueous solution of βCD

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and the suspension formed was subjected to stirring for 48 h upon heating at 50 °C to remove

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the excess ethanol. Then, they were subjected to the freeze-drying process to achieve the solid

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inclusion complex.

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Solid-state characterizations. Attenuated Total Reflection - Fourier Transform Infra-Red

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Spectroscpy (FTIR-ATR) spectra of IVER, EPRI, βCD, IVER/βCD, EPRI/βCD, and their

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respective physical mixtures (PMs) at a mole ratio of 1:1 were recorded between 4000 and

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400 cm–1 using a Perkin Elmer Spectrum TwoTM FTIR spectrometer and KBr pellets. The

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spectra were recorded as the average of 16 scans with a spectral resolution of 2 cm–1. Perkin

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Elmer Spectrum ES 192 software (version 10.03.08.0133) was used for the analysis of the

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spectra.

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TGA and DTA were performed using a thermobalance 2950 Thermal Analysis Instrument

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(TA Instrument, New Castle, DE, USA). Data were recorded for IVER, EPRI, βCD,

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IVER/βCD, EPRI/βCD, and their PMs at a mole ratio of 1:1. The experiments were carried

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out using an inert atmosphere (N2) of 50 mL min–1, a heating rate of 10 °C min–1, and

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sensitivity of 1.0 °C. For each experiment, approximately 3.5 mg of sample was used, which

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was placed in open alumina pans.

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ITC. ITC was performed in triplicate using a Microcal Microcalorimeter VP-ITC

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(Microcal Company, Northampton, MA, USA). The solutions were prepared by dissolution of


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IVER, EPRI, and βCD in DMSO:water mixture (9:1, v/v). Each titration experiment consisted

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of 51 successive injections of IVER or EPRI solution (30 mmol L-1) into the reaction cell

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charged with 1.5 mL of βCD solution (2.0 mmol L-1) at intervals of 540 s. The initial 1.0 µL

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injection was discarded from each dataset to eliminate the effect of titrant diffusion across the

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syringe tip during the preequilibration process. Subsequent injections were carried out using a

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constant volume of titrant (5.0 µL). A blank experiment was performed by injection of

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AVMs’ solution into the solvent. The concentration correction and integration of the heat

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flow peaks involved in partial molar enthalpy of βCD were made with software Microcal

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Origin 6.0 for ITC. The calculation of the binding constant (Kb), stoichiometry (N), and

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enthalpy of reaction (∆rH0) were performed using the one set of site model (Wiseman

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Isotherm) provided by the Microcal Origin 6.0 software.23 The Gibbs free energy (∆rG0) and

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entropy (T∆rS0) of interaction were calculated by the classical thermodynamic equations also

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provided by the same software.

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NMR experiments. Structural characterizations of IVER/βCD and EPRI/βCD complexes

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were performed in solution at mole ratio 1:1, using 1H NMR chemical shifts (δ) and a 2D-

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NOESY contour map, both obtained at 298.15 K in a Varian 400 MHz spectrometer using a

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probe of 5 mm 1H/X/D Broadband at 25 °C. The solutions of IVER, EPRI, βCD, IVER/βCD,

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and EPRI/βCD were prepared in DMSO-d6 (Cambridge Isotope Laboratories, Tewksbury,

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MA, USA) at concentrations of 0.1 mmol L-1. The DMSO-d6 signal at δ = 2.50 ppm was used

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as a reference. 2D-ROESY experiments were recorded at a spin lock of 600 ms. NMR data

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were all processed and analyzed using the MestReC NMR software (version 4.9.9.6).

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Computational details. Initially, the geometries for the isolated species IVER, EPRI, and

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βCD were fully optimized, without any geometrical or symmetry constraints, using DFT with

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the B97D24 functional with Pople’s standard split valence 6-31G(d,p) basis set.25 The

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B97D/6-31G(d,p) harmonic frequencies were obtained for the isolated species, identifying 7 ACS Paragon Plus Environment


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them as true minima on the potential energy surface. For each inclusion complex, three spatial

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orientations were assumed considering the 1:1 mole ratio: modes A, B, and C as described in

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Figure S2.

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Once the initial guess geometries for the complexes were obtained, all six structures (three

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for IVER/βCD plus three for EPRI/βCD) were also fully optimized at the B97D/6-31G(d,p)

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level of theory. The B97D harmonic frequencies characterized the six complexes’ geometries

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as true minima on the PES. Further, the B97D harmonic frequencies were also used to

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estimate the Gibbs free energy of reaction in the gaseous phase (∆G). Guided by our very

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recent experience, we decided to choose the dispersion-corrected functional B97D, which

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carries in its formalism the dispersion effects, which are important parameters for

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supramolecular systems.26–28

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The solvent effect (water) was taken into account by the solvation model based on

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density.29 In the aqueous medium, the solvent effect is substituted by its dielectric constant

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(water ε = 78.4). The solute is set in a cavity of adequate shape to enclose the entire molecule,

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which is rapidly covered in the continuum dielectric. All DFT calculations performed were

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done with the Gaussian 2009 quantum mechanical package.30

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Colloidal characterization of HNPs. DLS experiments were performed in a Malvern

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Zetasizer Nano ZS 90 particle analyzer (Malvern Instruments Ltd., Malvern, Worcestershire,

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UK) using square polyethylene cells to measure the average hydrodynamic diameter (Dh) of

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IVER, EPRI, IVER/βCD, and EPRI/βCD nanoprecipitates formed when DMSO solutions of

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these compounds were titrated in water.

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Concerning the very low aqueous solubilities of AVMs and their inclusion compounds,

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around micromolar, stock solution samples were prepared by initial dissolution of 1.0 mg of

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IVER, EPRI, or an equimolar amount of IVER/βCD or EPRI/βCD in 0.5 mL of DMSO.

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Subsequently, 25 injections of 20 µL of these DMSO solutions were gradually titrated in a


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greater volume of ultrapure water (2 mL) and the HNPs were spontaneously formed by simple

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mixture. These samples were submitted to a monochromatic light (4 mW He–Ne laser at 633

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nm) and the scattered light intensity was measured at an angle of 90°. The Dh were

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determined by the average of five independent measurements, each of them obtained as the

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mean of 30 counts.

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ZPs were also determined in the Malvern Zetasizer Nano ZS 90, by means of the laser

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Doppler microelectrophoresis technique, at a scattering angle of 173°,31 using a glass cuvette

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into which the measuring cell (Dip Cell) was immersed. ZP values were calculated as the

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average of 10 independent measurements, each of them obtained as the mean of 30 counts.

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The same procedure used in the hydrodynamic diameter experiments were used here.

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Larvicidal assay with the HNPs. Aedes aegypti eggs (PPCampos strains) were donated by

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the Departamento de Biologia Geral from the Universidade Federal de Viçosa, Minas Gerais

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State, Brazil. The eggs were immersed in dechlorinated water to help emergence of larvae,

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which were maintained in plastic basins at 26 ± 2 °C, RH > 70% and photoperiod 12L:12D.

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The water was changed in the system daily by replenishing the food until the individuals

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reached the fourth instar larval stage. After the individuals reached the pupal stage, they were

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discarded. The larvae were collected for use in the bioassay when they reached the desired

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instar (L1 or L4).

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Experiments involving first instar larvae of Aedes aegypti were performed using an

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adaptation of the method developed by Pridgeon et al.32 This method consists of a rapid

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screening test to identify the larvicidal potential of the compounds. Initially, the sample stock

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solutions were prepared by initial dissolution of IVER, EPRI, IVER/βCD, or EPRI/βCD in an

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appropriate volume of DMSO. Then, five first instar larvae ca. 24 h after egg hatching were

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added to 1.5 mL Eppendorf® tubes together with 940 µL of dechlorinated water and 10 µL of

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a food suspension. Thereafter, 50 µL of each DMSO stock solution was added to the



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Eppendorf® tube to produce the HNPs, and until all the suspensions reached the desired

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concentration of the compound and 1% (v/v) of DMSO. The concentrations used in the

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experiments were 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 µmol L-1. The mortality of the

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larvae was determined after 24 h of incubation at 26 ± 2 °C and RH > 70%. The larvae were

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considered dead when they did not respond to a stimulus or did not rise to the surface of the

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liquid, and three replicates were performed in three independent experiments.

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Experiments involving fourth instar larvae of Aedes aegypti were performed using an

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adaptation of the method developed by the World Health Organization (WHO).33 Briefly, 24

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instar larvae were added to a 250 mL beaker together with 50 mL of dechlorinated water and

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gold fish food. Thereafter, 50 mL of each sample stock solution (described above) was added

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to the beaker until the suspension reached the desired concentration of the compound and 1%

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(v/v) DMSO. The concentrations used in the experiments were 0.001, 0.01, 0.1, 1, 10, and

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100 µmol L–1. The mortality of the larvae was determined after 24 h and 48 h of incubation at

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26 ± 2 °C and RH > 70%. Three replicates were performed in three independent experiments.

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For both experiments, curves of the percentage mortality versus the logarithm of the

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compound concentration were constructed using the Origin 8.0 program (OriginLab,

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Northampton, MA). The lethal concentration value (LC50) was calculated by probit analysis

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using the same software.

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MTT assay. The cytotoxicity tests were performed using strains of WI26VA4 SV40-

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transformed human fibroblasts cells enriched with Eagle’s Minimum Essential Medium

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(EMEM). The cells were cultured for replication and, after reaching an appropriate

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confluence, were placed in 96-well plates (5.103 cells). The cells were maintained for 48 h at

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37 °C in an atmosphere containing 5% CO2 with EMEM supplemented with sodium

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bicarbonate, L-glutamine, and 10% (v/v) fetal bovine serum sterile. Each compound was

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added to microplate wells containing the cells at varying concentrations of 0.001 to 500 µmol


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L-1 to obtain a final volume of 100 µL. Then, the microplates were again incubated for 48 h at

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37 °C in an atmosphere containing 5% CO2. MTT was used to determine cell viability. Four

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replicates were performed in two independent experiments. The results were plotted

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according to the percentage of cell viability versus molar concentration of the sample using

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the software GraphPrism 6.0, and IC50 values were determined.

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Safety information. During the manipulation of chemicals products and AVMs is

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recommend avoiding the contact with the skin and mucosas using fume hoods, goggles and

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gloves. About the inclusion compounds, once they are new compounds it is recommended the

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same care from the AVMs. The survival larvae were filtered in filter paper and systematically

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eliminated before discard.

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RESULTS AND DISCUSSION

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Solid-state characterization. FTIR spectra of IVER, EPRI, βCD, IVER/βCD, EPRI/βCD,

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and their PMs are shown in Figure 1. They were used to verify the existence of intermolecular

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interactions between βCD and AVMs in the solid state. The βCD spectrum is in accordance

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with those previously described in the literature with the main absorptions at 3380, 2925,

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1158, 1082, 1028, and 942 cm–1, which correspond to ν(O–H), ν(C–H), δ(O–H), ν(C–C), and

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the skeletal vibration involving the α-1,4 linkage, respectively.34

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The IVER spectrum shows main absorptions centered at 3480, 2965, 1735, and 1341-1000

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cm–1, which correspond to ν(O–H), ν(C–H), ν(C=O), and symmetrical and asymmetrical

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stretching of the C–O–C group, respectively. For EPRI, the main bands observed were ν(O–

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H) at 3457 cm–1, ν(C–H) of CH and CH2 groups at 2969 cm–1, ν(C–H) of CH3 groups at 2933

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cm–1, ν(C=O) of the carbonyl group in the macrolide at 1735 cm–1, and C–O–C flexion of

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glycosidic and ether bonds in 1161 cm–1.35 Furthermore, bands centered at 1659 and 1544 cm–



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1

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group present only in EPRI, were observed.

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attributed to ν(C=O) of secondary amides, characteristic of the substituent acetylamino

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In the spectra of the PMs, no significant changes in the position of the main absorptions in

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the relation-free compounds were observed. The main differences in the FTIR profiles can be

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justified by superposition of the βCD and AVM bands.

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FTIR spectra of IVER/βCD and EPRI/βCD complexes showed significant changes in the

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profile of the bands. Overall, a sharp attenuation of vibration modes was observed in the

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range from 1800 to 1300 cm–1 due to the vibrational restriction of molecules upon inclusion

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or association with βCD. The sharpening of hydroxyl and amide bands at around 3400 cm–1

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(ν(OH) and ν(NH)) was ascribed as the formation of a new pattern of hydrogen bonding.

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Other bands of IVER and EPRI at 1735 cm–1 (ν(C=O) of carbonyls), 1678 cm–1 and 1635 cm–

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1

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of several levels of polarization of moieties upon inclusion.

(ν(C=C) of alkenes), and 1400–1300 cm–1 (δ(C–H) and δ(C–O–H)) were shifted as a result

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TGA and DTA are important analytical tools for characterizing the stability of complexes

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upon heating. Figure 2 shows the TGA and DTA curves for IVER, EPRI, βCD, their PMs,

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and inclusion compounds.

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The decomposition of pure βCD is well documented, and the data obtained in this study are

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in good agreement with the reported literature.36 The TGA curve for IVER shows a first mass

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event (∆m = 5.2%) in the range from 145 to 168 °C, attributed to the evaporation of residual

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synthesis solvents.37 The decomposition of IVER begins with a small mass loss event between

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193 and 280 °C (∆m = 10.6%) followed by successive events of mass loss until complete

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decomposition of the material. In the TGA curve for EPRI, an endothermic peak that finished

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at 95 °C was ascribed to dehydration of the sample. The first decomposition event occurs

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between 227 and 341 °C, being responsible for the higher mass loss (∆m = 63.1%). In the

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DTA curves for the two AVMs, these events were all observed as endothermic peaks. 12 ACS Paragon Plus Environment

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TGA and DTA curves for PMs did not overlap the inclusion complexes’ (IVER/βCD and

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EPRI/βCD) curves. This indicates that there are significant differences in the intermolecular

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interactions established in the inclusion compounds compared with the PMs. In DTA curves

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for IVER/βCD and EPRI/βCD, the dehydration temperature ranges are shifted to lower

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temperatures in relation to the free βCD, indicating that the CDs molecules establish weaker

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interactions with the remaining water molecules after inclusion.38 Furthermore, the

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decomposition events of the inclusion complex occurred at intermediate temperatures

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between that identified for the free AVMs and βCD, as a consequence of new intermolecular

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interactions established by inclusion. These data suggest a higher thermal stability for

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complexed AVMs compared with the free ones.

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ITC. ITC experiments were performed to determine thermodynamic parameters of

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complexation in solution. Figure 3 shows the titration curves for IVER/βCD and EPRI/βCD

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systems, after subtraction of the dilution curve. The calculated thermodynamic parameters of

309

binding (∆bG0, ∆bH0, T∆bS0), the binding constant (Kb), and the stoichiometric coefficient (N)

310

for the interaction of the AVMs with βCD are shown in Table 1.

311

In the systems, the stoichiometric coefficient was close to unity (N ≈ 1), indicating that the

312

1:1 stoichiometries are the most favored. Thus, it is justified to prepare the inclusion

313

compounds with this mole ratio. Moreover, the similar values of N for the two systems were

314

expected due to the structural similarity.

315

In both cases, the bindings were spontaneous (∆bG0 < 0), endothermic (∆bH0 > 0), and

316

favored by entropy (T∆bS0 > 0). Positive enthalpy values suggest a consumption of energy to

317

break down intermolecular interactions in supramolecular aggregates or solvation structures.

318

The positive values for the entropic component were attributed to the occurrence of

319

hydrophobic interactions, i.e., desolvation of the precursors with a concomitant gain of

320

rotational and translational degrees of freedom by molecules. 13 ACS Paragon Plus Environment


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321

The obtained Kb values showed the same magnitude for both systems, corroborating the

322

hypothesis about the structural similarity of the supramolecular complexes. Their values are

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moderate compared with other systems and similar to that found in the literature for inclusion

324

compounds.39 However, the higher Kb value obtained for the IVER/βCD system than the

325

EPRI/βCD system indicates a stronger interaction between molecules. This can be related to

326

the absence of the double bond and the acetylamino group in IVER, which contribute to a

327

greater hydrophobicity of IVER in relation to EPRI, favoring a greater affinity with βCD in

328

solution.

329

NMR. 1H and 2D-ROESY NMR techniques were used to obtain evidence about the

330

AVMs’ interaction modes with the hydrophobic cavity of βCD. Because of the structural

331

complexity of IVER and EPRI, the assignment of the 1H NMR signals of the free molecules

332

was performed using data from the literature.40,41 Table S1 presents the 1H NMR data of free

333

and complexed βCD, while Tables S2 and S3 present the 1H NMR data of the free and

334

complexed AVMs.

335

Tables S1–S3 show several changes in the chemical shifts of complexed βCD and AVMs

336

as the result of new intermolecular interactions. For the βCD, displacements of chemical

337

shifts of the OH(2) and OH(3) hydrogens located on its outer face and the OH(6) hydrogen

338

located in its narrower side were observed. It was not possible to determine the chemical shift

339

variations of the signals referring to the CH(3) and CH(5) hydrogens of the βCD due to the

340

overlapping of these signals with those of the AVMs. In both IVER/βCD and EPRI/βCD

341

systems, changes in the chemical shifts of βCD hydrogens were similar, suggesting the

342

establishment of intermolecular hydrogen bonds in a supramolecular assembly.

343

The 1H NMR data of AVMs showed changes in chemical shifts of the benzofuran ring

344

hydrogens (Tables S1–S3), suggesting the inclusion of this moiety in the CD cavity. In

345

addition, the deoxysugar L-oleandrose groups from AVMs have high rotational mobility,


Page 15 of 43


346

enabling their interaction with the external face of βCD. This could also explain the

347

displacement of the hydrogen signals in the external face of βCD on inclusion.

348

To improve our understanding of the topology of inclusion complexes, 2D-ROESY

349

experiments were performed, which showed cross-peak correlations at a short distance (less

350

than 5 Å) between AVMs’ hydrogens and those hydrogens from the βCD.42 Selected regions

351

of the 2D-ROESY contour maps for IVER/βCD and EPRI/βCD are shown in Figures 4A and

352

4B, respectively. Correlations between OH(2) and OH(3) hydrogens from βCD and OH(8a)

353

hydrogens from the benzofuran ring from IVER or EPRI were observed in these figures,

354

confirming the inclusion of this moiety in βCD. From these experiments, it was not possible

355

to find any other NOE correlation due to the overlap with other intramolecular correlations in

356

the same region.

357

Theoretical approach. The formation of an inclusion complex can be understood in terms

358

of the stabilization of parts or the entire guest inside the hydrophobic cavity of the CDs. From

359

the theoretical point of view, continuum solvation models can be used in quantum mechanical

360

studies of the inclusion process in the condensed phase. Table 2 contains the relative

361

interaction energy (∆∆E) and relative Gibbs free energy (∆∆G) evaluated at the B97D/6-

362

31G(d,p) level of theory for the inclusion process of IVER and EPRI in βCD calculated in the

363

aqueous phase.

364

Analyzing Table 2, based on ∆∆E and ∆∆G values, one can immediately observe that the

365

most stable inclusion complex among the three possible arrangements is mode A of inclusion,

366

both for IVER/βCD and EPRI/βCD complexes. The reason for such a considerable

367

stabilization observed for this complex can be explained by a meticulous structural analysis at

368

the molecular level, which revealed that two hydrogen bonds are established between IVER

369

and EPRI and the secondary hydroxyl group of the βCD (see Figure 5). These hydrogen

370

bonds may be considered the main forces responsible for the stabilization of the complex, 15 ACS Paragon Plus Environment


Page 16 of 43

371

which leads to the IVER/βCD and EPRI/βCD complex in modes being the most energetically

372

favored compared with the other two complexes in modes B and C.

373

Finally, the good overall agreement between the theoretical data and IR and NMR

374

experimental findings provides support for the presence and predominance of mode A

375

complexes in the experimental sample.

376

Colloidal stability of the HNPs. HNPs are solid insoluble compounds usually produced in

377

a mixture of solvents where water is the main component. They can be stabilized in an

378

aqueous environment by the appropriate concentration control of the solid phase and mixture

379

ratio of solvent/cosolvent. Figures S3 and S4 present the solubility curves of IVER,

380

IVER/βCD, EPRI, and EPRI/βCD. The great advantage of these materials is the possibility to

381

produce simple formulations without the need to use costly additives or surfactants, acting as

382

a controlled release system driven by dissolution of the solid phase.43–45

383

The hydrodynamic diameter (Dh) and ZP measurements were recorded to evaluate the

384

effect of βCD on the colloidal properties of HNPs produced in DMSO/water solution. Figures

385

6 and 7 show ZP and Dh measurements, respectively, for IVER, EPRI, IVER/βCD, and

386

EPRI/βCD obtained by titration.

387

All systems (IVER, IVER/βCD, EPRI, and EPRI/βCD) produced HNPs in DMSO/water

388

mixture with negative ZP values ranging from –20 to –10 mV, which was attributed to the

389

partial ionization of acidic OH groups. The low ZP values determined for the systems suggest

390

the tendency to flocculate.46,47 Contrary to that observed for the IVER/βCD system,

391

EPRI/βCD nanoprecipitates showed more negative ZP values at concentrations lower than

392

150 µmol L-1, indicating a greater colloidal stability in this range. Its behavior was ascribed to

393

the presence of an amide group in the EPRI molecular structure, which favors the formation

394

of hydrogen bonds with water, thereby increasing its colloidal stability.


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395

For the two systems, the increasing concentration of the compounds in suspension leads to

396

an increase in Dh value, because of a gradual increase of the particle–particle interactions. For

397

free IVER, high polydispersity is observed above the concentration of ≈ 300 mmol L-1, where

398

hydrophobic interactions can cause coalescence of particles and inhomogeneity of size.

399

The inclusion compounds exhibited lower sizes than free AVMs. This can be justified by

400

the higher interaction of inclusion compounds with water due to the presence of hydroxyls of

401

βCD and its abilities to form hydrogen bonding or ionize, reducing therefore the occurrence

402

of hydrophobic interactions. In the presence of βCD, the reduction of size is much more

403

pronounced for the IVER/βCD system, which matches with the stronger affinity between the

404

molecules, as shown by ITC experiments.

405

Comparing the results for the free and complexated IVER and EPRI HNPs, lower Dh

406

values were observed for the second one, which was attributed to the greater solubility (lower

407

hydrophobicity), provided by the presence of the polar acetylamino group and absence of

408

ethyl group.

409

Larvicidal assay. Pridgeon et al. proposed a rapid method for the evaluation of potential

410

compounds with larvicidal activity using mosquito larvae in the first stage of development.32

411

It permits the testing of a large number of compounds in various concentrations with a small

412

number of larvae (five larvae per test). The use of first instar larvae reduces the time, labor,

413

and care required to create the larvae. In contrast, the WHO recommends performing the test

414

using the third and fourth instar larvae33 because these forms are more resistant than first and

415

second forms and because the pupal stage does not feed. In this work, the larvicidal activity of

416

IVER/βCD and EPRI/βCD HNPs (and their precursors) on first and fourth instar larvae of

417

Aedes aegypti was evaluated to verify the relation of the toxicity of the compounds to the

418

development of larvae (Figures S5–S7). The HNPs were formed by the addition of larvicides’

419

DMSO solution in water, as described in the experimental section.



Page 18 of 43

420

Table 3 shows the lethal concentration values at 50% of the larvae (LC50 in µmol L–1 and

421

ppm) for IVER, EPRI, and their inclusion compounds (IVER/βCD and EPRI/βCD) against

422

first and fourth instar larvae of Aedes aegypti. The dechlorinated water and 1% DMSO were

423

used as a control and they showed the percentage of mortality to be in the 2–7% and 6–9%

424

range, respectively. βCD was inactive in concentrations tested (

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