SUPPORTING INFORMATION. Zein nanoparticles as eco-friendly carrier systems for botanical repellents aiming sustainable agriculture

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1 SUPPORTING INFORMATION Zein nanoparticles as eco-friendly carrier systems for botanical repellents aiming sustainable agriculture Jhones L. de Oliveira a, Estefânia V.R. Campos b, Anderson E.S. Pereira b, Tatiane Pasquoto c, Renata Lima c, Renato Grillo d, Daniel Junior de Andrade e, Fabiano Aparecido dos Santos e, Leonardo Fernandes Fraceto* a,b a São Paulo State University (UNESP), Institute of Science and Technology, Sorocaba, SP, Brazil b Department of Biochemistry, Institute of Biology, State University of Campinas, Campinas, SP, Brazil c Department of Biotechnology, University of Sorocaba, Sorocaba, Brazil d São Paulo State University (UNESP), Department of Physics and Chemistry, School of Engineering, Ilha Solteira, SP, Brazil. e São Paulo State University (UNESP), College of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil Corresponding author: leonardo@sorocaba.unesp.br (L.F.F.) 1

2 1. Methodology validation for botanical compounds Quantification of the botanical repellents was performed by high performance liquid chromatography, employing an UltiMate 3000 system (Thermo Scientific). Chromeleon 7.2 software was used for acquisition and interpretation of the chromatograms. The geraniol analysis was performed using a Phenomenex Gemini C 18 reverse phase column (150 mm 4.6 mm, 5.0 µm) kept at 30 C. The mobile phase was acetonitrile:water (60:40, v/v), at a flow rate of 1 ml/min. The injection volume was 100 µl and the detector wavelength was set at 210 nm. The analysis of R-citronellal was performed using a Phenomenex Kinetex C 18 reverse phase column (150 mm 4.6 mm, 3.0 µm) kept at 30 C. The mobile phase was acetonitrile:water (70:30, v/v), at a flow rate of 1 ml/min. The injection volume was 100 µl and the detection wavelength was 210 nm. All the analytical curves showed correlation coefficients (r 2 ) higher than The detection limits (DLs) for GRL and R-CTL were 0.39 ± 0.01 and 3.37 ± 0.12 µg/ml, respectively. The quantification limits (QLs) were 1.30 ± 0.09 and 5.61 ± 0.10 for GRL and R-CTL, respectively. 2. Fourier Transform Infrared Spectroscopy analysis The spectrum of zein (Figure S2-A) showed bands between 3100 and 2800 cm -1, attributed to C-H groups of fatty acids and amino acids. Intense bands between 1800 and 1500 cm -1 were assigned to amides I and II, and bands from 1300 to 1500 cm -1 were attributed to stretching of =CH 2 and C-H bonds of lipids and proteins. The spectrum of Pluronic F-68 (Figure S2-B) showed a band at 2884 cm -1, assigned to stretching vibrations of C-H groups, and bands from 948 to 1110 cm -1, associated with symmetric and asymmetric stretching vibrations of C-O of ether groups. The spectrum of geraniol (Figure S2-C) showed bands associated with alcohol -OH at 3326 cm -1, alkane C-H vibration at 1669 cm -1, and C=C bond vibration at 1010 cm -1. The R-CTL spectrum (Figure S2-D) showed a band between 2900 and 3000 cm -1 corresponding to the stretching of aliphatic C-H bonds, a band at 2700 cm -1 assigned to C-H stretching, and a strong band at 1740 cm -1 (C=O) confirming the presence of the aldehyde group. Figure S2-E shows the infrared spectrum for the control zein nanoparticles (without AIs). A signal at 3340 cm -1, indicative of the O-H group, was probably associated with 2

3 the presence of water. Also present were bands associated with the characteristic groups of the zein protein, such as amides I and II. The spectrum of the zein nanoparticles loaded with geraniol (Figure S2-F) showed characteristic peaks of zein (amides I and II). An intense peak at around 3326 cm -1 was attributed to the alcohol groups of GRL. Shifts of the bands corresponding to the bonds -C-H (at 2967 and 2916 cm -1 ) and C-O- (at 1010 cm -1 ) were also observed, probably due to interactions with the repellent. Figure S2-G shows the spectra for zein nanoparticles loaded with R-CTL, with bands corresponding to the presence of water (O-H signal at 3340 cm -1 ), amides I and II of zein, and shifts of the aliphatic C-H bond stretching bands, indicative of interactions with the botanical repellents. 3

4 Figure S1: Stability of botanical repellents as a function of time. A significance level of p < 0.05 was considered for the differences obtained within the same group; a*, b*, c*, d*, and e* indicate significant differences relative to days 0, 15, 30, 60, and 90, respectively. 4

5 Figure S2: FTIR spectrum: A- zein powder; B- Pluronic F-68 surfactant; C- GRL; D- R-CTL; E- NP_Z; F- NP_GRL and G- NP_R-CTL. 5

6 Figure S3: Effect of different formulations under fresh mass and dry mass of beans seedlings (P. vulgaris), after seed treatment. A) Fresh mass; B) Dry mass. Being: Zein nanoparticles (NP); Pluronic F-68 surfactant; Geraniol emulsified with surfactant (GRL + Pluronic 1%); zein nanoparticles loaded with geraniol (NP_GRL); R-Citronellal emulsified with surfactant (R-CTL + Pluronic 1%); Zein nanoparticles loaded with R- citronellal (NP_R-CTL). A significant difference of p <0.05 was considered for statistical differences observed between the groups of the same concentration, where a * represents significant variations between the groups in relation to the control. A) Efeito de diferentes formulações sob a massa fresca e massa seca de plântulas de feijão (Phaseolus vulgaris), após o tratamento das sementes. A) Massa Fresca; B) Massa seca. Sendo: Nanopartículas de zeína (NP); Tensoativo Pluronic F-68; Geraniol emulsionado com tensoativo (GRL + Pluronic 1%); nanopartículas zeína carregadas com geraniol (NP_GRL); R-Citronelal emulsionado com tensoativo (R-CTL + Pluronic 1%); Nanopartículas de zeína carregadas com R-citronelal (NP_R-CTL).. Uma diferença significativa de p <0,05, foi considerada para diferenças estatísticas observadas entre os grupos da mesma concentração, onde a * representa variações significativas entre os grupos em relação ao controle. B) 6

7 Figure S4: Repellent activity of zein nanoparticles without botanical repellents and the Pluronic F-68 surfactant against two-spotted spider mite (Tetranychus urticae). A) Concentration of 5 mg/ml; B) Concentration of 0.5 mg/ml and C) Concentration of 0.05 mg/ml. 7