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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Engineering Atrazine Loaded Poly (lactic-co-glycolic Acid) Nanoparticles to Ameliorate Environmental Challenges Brian Schnoor,†,‡ Ahmad Elhendawy,†,‡ Suzanna Joseph,†,‡ Mark Putman,†,‡ Randall Chacoń -Cerdas,§ Dora Flores-Mora,§ Felipe Bravo-Moraga,∥ Fernando Gonzalez-Nilo,∥ and Carolina Salvador-Morales*,†,‡ †

Bioengineering Department, George Mason University, 4400 University Drive MS 1J7, Fairfax, Virginia 22030, United States Institute of Advanced Biomedical Research, George Mason University, 10920 George Mason Circle, MS1A9, Manassas, Virginia 20110, United States § InstitutoTecnológico de Costa Rica, Biotechnology Research Center, Cartago, Costa Rica ∥ Center for Bioinformatics and Integrative Biology, Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago 8370146, Chile

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ABSTRACT: The use of herbicides plays a vital role in controlling weeds and conserving crops; however, its usage generates both environmental and economic problems. For example, herbicides pose a financial issue as farmers must apply large quantities to protect crops due to absorption rates of less than 0.1%. Therefore, there is a great need for the development of new methods to mitigate these issues. Here, we report for the first time the synthesis of poly(lactic-co-glycolic-acid) (PLGA) nanoherbicides loaded with atrazine as an active ingredient. We used potato plants as a biological model to assess the herbicidal activity of the engineered PLGA nanoherbicides. Our method produced nanoherbicides with an average size of 110 ± 10 nm prior to lyophilization. Fifty percent of the loaded atrazine in the PLGA matrix is released in 72 h. Furthermore, we performed Monte Carlo simulations to determine the chemical interaction among atrazine, PLGA, and the solvent system. One of the most significant outcomes of these simulations was to find the formation of a hydrogen bond of 1.9 Å between PLGA and atrazine, which makes this interaction very stable. Our in vitro findings showed that as atrazine concentration is increased in PLGA nanoparticles, potato plants undergo a significant decrease in stem length, root length, fresh weight, dry weight, and the number of leaves, with root length being the most affected. These experimental results suggest the herbicidal effectiveness of atrazineloaded PLGA nanoherbicides in inhibiting the growth of the potato plant. Hence, we present the proof-of-concept for using PLGA nanoherbicides as an alternative method for inhibiting weed growth. Future studies will involve a deep understanding of the mechanism of plant−nanoherbicide interaction as well as the role of PLGA as a growth potentiator. KEYWORDS: environmental technology, nanoherbicides, poly(lactic-co-glycolic-acid) (PLGA), atrazine, polymers, nanoparticles

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INTRODUCTION Protecting crops from nutrient stealing weeds is an essential part of agriculture. Herbicides play a vital role in controlling weeds and conserving crops. However, the use of herbicides generates many environmental and economic problems. Current herbicides pose a significant risk to the environment since vast quantities of herbicides are washed into streams and rivers as runoff, which can kill nontarget organisms and disrupt ecosystems.1−4 The widespread use of herbicides, such as atrazine, also presents economic problems because these herbicides evaporate quickly and are readily trapped in the top layer of soil due to soil absorption.5,6 Therefore, less than 0.1% of the applied herbicides reach the target organisms.7 Even the herbicide that reaches the target weed can be ineffective due to poor translocation in the weed and the development of herbicide resistant weeds. Because of this phenomenon, larger amounts of herbicides are required, which can exacerbate environmental damage. To overcome these problems, a new delivery system is needed to protect the herbicides, ensure that they reach the weed, and improve transport of the herbicide within the target weed. © XXXX American Chemical Society

Atrazine is the second most popular herbicide in the United States since it is very efficient in controlling weeds and has proven to be harmless toward corn crops.8 Nevertheless, atrazine can cause severe environmental damage. For instance, in the United States, it contaminates more water sources than any other pesticide.9 In the European Union, atrazine has already been banned because atrazine contamination in the groundwater exceeded the maximum limits set by law.10 Furthermore, studies indicated that atrazine has harmful effects on nontarget organisms in aquatic ecosystems.3,4,11 Nanoparticle-based delivery systems for herbicides also known as “nanoherbicides” have shown great promise to improve herbicidal efficacy.8,12−14 Nanoherbicides consist of a traditional herbicide encapsulated within a nanoparticle’s core, which protects and directs the herbicide to the target organism. Nanoherbicides have the potential to prevent the fast Received: April 14, 2018 Revised: June 14, 2018 Accepted: July 13, 2018

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DOI: 10.1021/acs.jafc.8b01911 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

formed into lactic and glycolic acid, respectively, which are metabolized by the Kreb’s cycle. The byproducts of the Kreb’s cycle are carbon dioxide and water. Therefore, PLGA is not toxic to humans, plants, or animals.19 Additionally, it is worth mentioning that PLGA can currently be purchased in kilograms or tons at low cost from Chinese manufacturers. Thus, the scaled-up production of PLGA nanoherbicides may not represent a technical challenge for the use of PLGA nanoherbicides in agricultural settings.

evaporation of naked herbicides and improve both the absorption of herbicides through the plant root and translocation within the plant.8,12−14 Furthermore, nanoherbicides can reduce the amount of herbicide trapped in the top layer of soil, consequently diminishing water contamination due to runoff removed from surface soil. Similarly, PEGylation of the nanoparticles’ surface can also improve absorption and translocation of the nanoparticles in the plant and decrease the amount of free herbicide that could contaminate streams and reach nontarget organisms. Thus, we can reduce the amount of herbicide delivered to nontarget species in surrounding or downstream areas by increasing the efficiency of delivering the herbicide to the target weed. Several research groups have synthesized a wide variety of nanoherbicides that combine surfactants, polymers, and metallic nanoparticles to facilitate the application of herbicides in crop fields by overcoming the insolubility of some herbicides. Each type of nanoherbicide has a specific purpose. For example, microemulsion (6−50 nm), nanoemulsion (20− 200 nm), and nanodispersion (50−200 nm) aim to increase the solubility of poorly water-soluble active ingredients (AI).14 There are other types of polymer-based nanoherbicides that seek to release the payload in a slow, sustained fashion while preventing premature degradation of the active ingredient. This class of nanoherbicides includes polymers such as poly(epsilon-caprolactone) (PCL),8,15,16 chitosan,17 and alginate.18 Here, we report a novel nanoherbicide delivery system based on a PLGA polymer. PLGA is a biocompatible, biodegradable, and Food and Drug Administration (FDA) approved copolymer formed by two different monomers: lactide and glycolide. PLGA provides several significant advantages over other polymer-based nanoherbicides. First, the degradation rate of this copolymer can be manipulated according to our agricultural needs by varying the ratio between lactide and glycolide monomers. The degradation of PLGA occurs via hydrolysis, which cleaves the ester bonds of the lactide and glycolide monomers. The ester bonds of the lactide monomers undergo a much slower degradation rate due to the steric effect of the CH3 group that is present in the lactide but not in the glycolide monomer.19 This chemical feature allows the tuning of the degradation profile of the PLGA nanoherbicide, which cannot be achieved with the PCL homopolymer. Moreover, chitosan, a highly hydrophilic polymer17 cannot be used to successfully encapsulate atrazine. Furthermore, PLGA has a degradation-based release profile (i.e., the active ingredient is released in unison with the degradation of the nanoparticle). This will prevent the problem of rapid separation of the active ingredient from the nanocarrier after reaching the soil/plant surface, which can significantly affect the transport of the active ingredient.20−22 Indeed, PCL also follows this degradationbased release profile and achieves a similar release effect. However, PCL presents its own environmental challenge due to its slow degradation rate. Unlike PLGA, which will completely degrade within 6 months at the most, PCL degrades in 1−3 years.23 This is an extended period of time for PCL to interact with organisms in the environment. Additionally, other polymers such as alginate are nontoxic only when in high purity. Obtaining that level of purity often involves extensive and expensive synthesis processes.24 PLGA, however, is a harmless polymer that degrades completely into nontoxic byproducts. In fact, when hydrolysis takes place, the lactide and glycolide monomers are trans-

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MATERIALS AND METHODS

Materials. The reagents atrazine, terbuthylazine, acetone, acetonitrile, formic acid, and ethanol were purchased from SigmaAldrich. Lecithin was acquired from Alfa Aesar, and PLGA was obtained from Lactel Absorbable Polymers. 1,2-Distearoyl-snglycero3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-NH2) was purchased from Laysan Bio Inc. Uranyl acetate and the TEM copper mesh grids were acquired from Electron Microscopy Sciences. Synthesis of PLGA Nanoherbicides. PLGA nanoherbicides were synthesized using a modified nanoprecipitation method developed in-house. Briefly, we prepared stock solutions of 1 mg/ mL DSPE-PEG-NH2 and 1 mg/mL Lecithin in 4% ethanol. 900 μL of the DSPE-PEG-NH2 solution and 1200 μL of the lecithin solution were added to a 20 mL glass vial containing 12 mL of deionized water (diH2O) and 1700 μL of 4% ethanol. The solution was then heated to 68 °C for 5 min under light stirring using an IKA hot plate. A stock solution of 2 mg/mL of PLGA in acetone was prepared. Subsequently, 1 mL of the PLGA solution and 2 mg of atrazine were added to a smaller glass vial. Using a 25 G 1 × 1.5 needle and plastic syringe, the organic phase was added dropwise to the aqueous phase under moderate stirring. The needle was submerged into solution and slowly moved up and down as the organic phase was added to the aqueous phase to help prevent particle aggregation. To synthesize larger quantities of nanoherbicides, this procedure was scaled up 5-fold. To accomplish this task, the same stock solutions were used to prepare an aqueous solution consisting of 60 mL of diH2O, 4.5 mL of DSPE-PEG-NH2 stock solution, and 6 mL of lecithin stock solution. This aqueous phase was then heated to 68 °C for 8 min under light stirring. Then, 5 mL of PLGA stock solution was added to a glass vial containing 10 mg of atrazine. The organic solution was then added to the aqueous solution using the same method described above. Characterization of PLGA Nanoherbicides. Particle Size of PLGA Nanoherbicides. The nanoherbicide size was determined by the dynamic light scattering (DLS) technique. The DLS measurements were taken with an N5 Submicron Particle Size Analyzer (Beckman Coulter). DLS was first calibrated using standardized polymer beads of 100 nm in size. Then, 3 μL of the nanoherbicide solution was mixed with 2 mL of diH2O in a plastic cuvette. The cuvette was then placed in the DLS for analysis at 25 °C and 90° deflection. Morphology of PLGA Nanoherbicides. The morphology of PLGA nanoherbicides was examined by a transmission electron microscope (TEM). These samples were prepared by pipetting 10 μL of the nanoherbicide sample onto the carbon side of a copper grid. Then 10 μL of uranyl acetate was added to the copper grid, which was then allowed to dry overnight. Next, the samples were examined with the Tecnai G spirit BioTwin at 80 kV. Images were recorded with an AMT 2k CCD camera. Fourier Transform-Infrared (FT-IR) Spectroscopy Characterization. The PLGA nanoherbicides were characterized with a Jasco FT/IR 4100 spectrophotometer, operated in the range 550−4000 cm−1 employing 36 scans per sample and a resolution of 4 cm−1. The FT-IR measurements were performed on nanoherbicides (i.e., atrazine-loaded PLGA nanoparticles), unloaded PLGA nanoparticles (i.e., PLGA polymeric particles without atrazine) and atrazine. For each FT-IR spectrum, a sample of 2 mg was mixed with 200 mg of sodium bromide using a mortar and pestle before being compressed B


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Journal of Agricultural and Food Chemistry into a flat translucent disk. Subsequently, each sample was analyzed using an FT/IR spectrophotometer, and peaks were identified. Encapsulation Efficiency of PLGA Nanoherbicides. The percentage of atrazine encapsulated in the PLGA polymer matrix was determined using a liquid chromatography/mass spectrometry (LC-MS) technique. After nanoherbicide synthesis, we degraded the nanoherbicides with acetonitrile. One milliliter of acetonitrile was added to the nanoherbicide solution until it was perfectly clear. Next, the resulting solution was vortexed for 3 min. Subsequently, the solution was centrifuged in an Amicon filter with 100 kDa molecular cutoff at 2000 rpm, allowing the atrazine to pass into the filtrate. The filtrate was then protected from light exposure to prevent atrazine degradation. These samples were analyzed with a LC-MS instrument. Internal Calibration Standards. We first had to create a calibration curve for atrazine to analyze the atrazine concentration using the LC-MS technique. To accomplish this task, we prepared an atrazine stock solution of 1 mg/mL atrazine in methanol. Then, we prepared a stock solution of terbuthylazine (1 mg/mL) in acetone. An acetonitrile mobile phase was prepared by mixing 99 mL of high performance liquid chromatography (HPLC) grade acetonitrile and 1 mL of formic acid to produce 1% formic acid in acetonitrile. A diH2O mobile phase was also prepared by mixing 1 mL of formic acid and 99 mL of diH2O. Then, the complete mobile phase was prepared by mixing 50 mL of acetonitrile mobile phase and 50 mL of diH2O mobile phase. Standard 1 was prepared by adding 75 μL of terbuthylazine stock solution, 7.5 μL of atrazine stock solution, and 1417.5 μL of complete mobile phase to an HPLC vial to produce a 5 ng/μL atrazine concentration. Standard 2 was prepared using 75 μL of terbuthylazine stock solution, 22.5 μL of atrazine stock solution, and 1402.5 μL of mobile phase to obtain an atrazine concentration of 15 ng/μL. Standard 3 was prepared using 75 μL of terbuthylazine stock solution, 50 μL of atrazine stock solution, and 1375 μL of mobile phase to obtain a 33.33 ng/μL atrazine concentration. Standard 4 was prepared using 75 μL of terbuthylazine stock solution, 67.5 μL of atrazine stock solution, and 1357.5 μL of mobile phase to produce an atrazine concentration of 45 ng/μL. Finally, standard 5 was prepared using 75 μL of terbuthylazine stock solution, 100 μL of atrazine stock solution, and 1325 μL of mobile phase to obtain a 66.67 ng/μL atrazine concentration. These five internal standards were diluted 1000-fold in acetonitrile and analyzed with LC-MS using 1% formic acid in acetonitrile mobile solvent. The internal standards are used to create a calibration curve. The nanoherbicide sample for each time point, taken in triplicate, was diluted by mixing 300 μL of the nanoherbicide sample with 75 μL of the terbuthylazine internal standard prepared above and 1125 μL of complete mobile phase. This mixture resulted in a 5-fold dilution of the degraded nanoherbicide sample. The samples were further diluted 1000-fold in acetonitrile. Then, the diluted solution was analyzed using the LC-MS instrument and measured against the calibration curve created from the standard samples. We determined the concentration of atrazine in each nanoherbicide sample based on this calibration curve. Molecular Interaction Characterization among PLGA, Atrazine, and Solvents. We performed a configurational sampling using a Monte Carlo strategy to study the chemical interaction among PLGA, atrazine, acetonitrile, and acetone as implemented in Avila et al.25 The energy interaction analysis between the pairs of PLGA and the various molecules (e.g., atrazine, acetonitrile, and acetone) was performed using a total of 200,000 pair configurations. The energy interaction for each pair configuration was computed using MOPAC 2016 and the semiempiric PM7 method.26,27 The atrazine, acetonitrile, and acetone molecules were geometrically optimized using Gaussian 0928 with an HF/6-31G level of theory. Atrazine Release Profile of PLGA Nanoherbicides. The PLGA nanoparticles’ atrazine release profile was determined using dialysis along with the LC-MS technique to determine the percent of the encapsulated atrazine released over time. According to the nanoherbicide synthesis described above, 100 μL of the nanoherbicide solution was added to 6 mini-dialysis tubes with a pore size of 3600 Da. Then, these dialysis tubes were placed in a 2 L reservoir of

deionized water and stirred with a magnetic stirrer. Then, the minidialysis tubes were removed from the dialysis reservoir at 6, 12, 24, 36, 48, and 72 h time points. The volume contained in the minidialysis tubes at each time point was transferred to a glass vial, and an equal volume of acetonitrile was then added and vortexed to degrade the nanoparticles. All sample solutions were then diluted to 1050 μL using acetonitrile. This was repeated 3 times to obtain triplicate samples for each time point. The concentration of atrazine in each of these samples was then determined using an LC-MS instrument. Lyophilization Process of PLGA Nanoherbicides. The lyophilization process plays a key role in nanoherbicide synthesis to prevent PLGA degradation and facilitate the storage of nanoherbicides until their use. Several cryoprotectants, including sucrose and various polymers at different concentrations, were tested. Five percent of sucrose was the best cryoprotectant found because it greatly prevented the enlargement of the nanoparticle size. Thus, 5% sucrose was mixed with the filtered nanoherbicide solution in a 1 to 1 ratio before the solution was lyophilized. After lyophilization, the nanoherbicides were then resuspended to test the nanoherbicides’ solubility. In Vitro Plant Studies to Assess the Herbicide Activity of Nanoherbicides. We used potato plants as a biological model to assess the herbicidal activity of the engineered nanoherbicides (Biotechnology Research Center, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica). The in vitro plants were reproduced in complete culture medium following the protocol described in Flores et al.29 The plants were inoculated in microcuttings, three for each container. Twenty milliliters of complete culture medium was used per container. They were incubated at 22 °C in a controlled temperature environment with a photoperiod of 16/8 h. for a period of 22−30 days. Assessment of the Growth and Development of the Target Species Using an in Vitro Model. We assessed in vitro the effect of free herbicide and encapsulated herbicide in the PLGA matrix in plants with respect to the growth and development of the target organism (i.e., potato plant) using different atrazine concentrations. We used atrazine with 98% purity, and the nanoherbicides were composed of 42.38% atrazine and 57.62% PLGA, and lyophilized with 5% sucrose. Unloaded PLGA nanoparticles and the targeted organism cultured only with complete medium were the control experiments of this study. The concentrations of PLGA nanoparticles, atrazine, and nanoherbicides used in this study are shown in Table 1. The

Table 1. Targeted Organism (Potato Plant) Treated with Complete Medium, Atrazine, Nanoherbicides, and PLGA Nanoparticles at Different Concentrations treatment 0. 1. 2. 3. 4. 5. 6. 7. 8. 9.

control atrazine atrazine atrazine nanoherbicide nanoherbicide nanoherbicide PLGA nanoparticle PLGA nanoparticle PLGA nanoparticle

concentration 0.0 μg/mL 0.7 μg/mL 6.3 μg/mL 54.0 μg/mL 1.7 μg/mL 14.9 μg/mL 127.4 μg/mL 1.0 μg/mL 8.6 μg/mL 73.4 μg/mL

concentrations of nanoherbicide used were selected to account for the mass of the nanoparticle shell and administer the same amount of atrazine as the pure atrazine samples. Similarly, the concentrations of PLGA particles without atrazine were selected to administer the same amount of PLGA as the nanoherbicide trials. In the in vitro studies, we evaluated the following key variables: stem length, longest root length, number of leaves, and fresh and dry weight. We conducted these experiments in 50 replicates per treatment. C


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Journal of Agricultural and Food Chemistry Determination of Altered Cellular Division. We selected the root apex of each target species treated with PLGA, nanoherbicides, and atrazine less than 2 cm in length, and we added acetic acid/ ethanol (3:1) for 3 days, followed by three washes with diH2O. Then, we placed the treated apex in a solution of HCl of 0.1 M for 30 min. Finally, we washed the samples three times with diH2O. We took part of the root tip to analyze with the light microscope to investigate if the treatment produced morphological cellular alterations. We labeled this sample with Giemsa (3:1). We placed the samples on a microscope slide, and we added a drop of the dye. Then, we placed a coverslip on the top of the sample. Statistical Analysis. The data were analyzed using the ANOVA Welch and GAMES-Howell tests. All the tests were analyzed within the 95% confident interval, p = 0.05, using the statistical program Minitab 17.30

glycolide monomers to release the herbicide in a controlled release fashion over a relatively short period of time. Transmission Electron Microscopy and Dynamic Light Scattering. TEM revealed that PLGA nanoherbicides have a very well-defined spherical shape (Figure 2A). DLS results show that the average size of nanoherbicides in diH2O is 110 ± 10 nm before lyophilization (Figure 2B). These nanoherbicides remained in suspension for several weeks after their synthesis. We lyophilized the nanoherbicides after their synthesis to conduct in vitro plant studies. Before the lyophilization process, we incorporated 5% sucrose in the nanoherbicide solution to avoid precipitation after the freezedrying step. However, we observed a substantial increment in the nanoherbicide size after the lyophilization process. The size of the freeze-dried nanoherbicides is about 500 nm ±10 nm (Figures S1,S2). Subsequently, we tested the stability of these nanoherbicides in diH2O after the lyophilization process, and we found that they remained in suspension for several weeks. Molecular Interaction Characterization among PLGA, Atrazine, and Solvents. The encapsulation efficiency for a 5fold scaled-up prelyophilized nanoherbicide sample is 50%. Infrared studies demonstrated the association between atrazine and the polymeric chains of PLGA as shown in Figure 2C. The IR of atrazine shows a band at 3258 cm−1 which corresponds to stretching of the N−H bond present in the amine functional group of atrazine, whereas the band at 2973 cm−1 is associated with the stretching of the alkyl group C−H bond. The bands at 1622 and 1557 cm−1 indicated the deformation of CC and CN bonds, respectively, in atrazine. PLGA polymeric nanoparticles (e.g., unloaded nanoherbicides) showed bands at 3388 cm−1 (O−H bond stretching), 1759 cm−1 (stretching of the carbonyl CO bond), and 1068 cm−1 (angular deformation of C−O). The IR spectrum of PLGA nanoherbicides (e.g., atrazine-loaded PLGA nanoparticles) showed three distinctive peaks at 3389 cm−1, 1760 cm−1, and 1069 cm1 that correspond to the PLGA component of the nanoherbicide sample, while the peaks at 1623 and 1555 cm−1 correspond to atrazine’s bands. Thus, these peaks indicate the presence of atrazine in the polymeric matrix of the nanoherbicide. Grillo et al. achieved between 80% to 90% atrazine encapsulation efficiency using an interfacial deposition of preformed polymer method and PCL as a polymer matrix.16 This process produced atrazine-loaded nanoherbicides of approximately 200−300 nm size before lyophilization. Although our modified nanoprecipitation method provides atrazine-loaded PLGA nanoparticles with lower encapsulation efficiency, the particle size of our prelyophilized nanoherbicide sample is 110 ± 10 nm. Pereira et al. reported the encapsulation efficiency of PCL nanocapsules and nanospheres of 93%.8 The size of the nanocapsules and nanospheres was 513 ± 7.5 nm and 365.1 ± 0.16 nm, respectively. Thus, it seems that the encapsulation efficiency is related to the particle’s size. The higher the particle size, the higher is the encapsulation efficiency. To further investigate the parameters that affect the nanoherbicides’ encapsulation efficiency, we performed quantum mechanics calculations to determine the type of interaction energy that takes place among PLGA, atrazine, and solvents (e.g., acetonitrile and acetone) as well as the magnitude of such interaction (Figure 2D). It is important to mention that we used acetone to dissolve PLGA during the nanoprecipitation method as described in the PLGA nanoherbicides’ synthesis. We only used acetonitrile as a solvent to

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RESULTS AND DISCUSSION PLGA Nanoherbicide’s Design. In the core−shell structure of the nanoherbicide, PLGA forms the core, and the shell is made of DSPE-PEG-NH2. Herbicides such as atrazine are embedded in the PLGA polymer matrix (Figure 1).

Figure 1. Nanoherbicide’s design. Nanoherbicides are formed by a self-assembly process using a modified nanoprecipitation method developed in-house. The nanoherbicide is composed of, lecithin, lipidPEG-NH2 and PLGA. Atrazine is encapsulated in the polymeric matrix.

Characterization of PLGA Nanoherbicides. Nanoherbicides have been synthesized with different polymers such as PCL and chitosan. To the best of our knowledge PLGA nanoherbicides have not been synthesized yet. Thus, we are the first research group reporting their synthesis, characterization, and herbicidal activity. The use of PLGA in agricultural settings has not been yet explored. We realized the great benefits that this polymer could have offered to agriculture because of its unique physicochemical properties. For example, since PLGA is a copolymer, we can fine-tune the degradation characteristics of PLGA nanoherbicides by varying the copolymer ratio. This specific functionality cannot be completely achieved by other polymers such as PCL and chitosan because they are not copolymers. Also, the fact that PLGA is a copolymer allows it to encapsulate nonwater-soluble herbicides such as atrazine. In this study, we used a 75:25 copolymer ratio, 75% of lactide, and 25% of glycolide monomers because this copolymer ratio makes the entire PLGA matrix more hydrophobic due to the steric effects given by the methyl group present in the lactide monomer. Atrazine can then be encapsulated in the polymer matrix. Additionally, the ratio in PLGA (75:25) still maintains enough hydrophilic D


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Figure 2. Characterization of PLGA nanoherbicides. (A) TEM image of nanoherbicides’ morphology. (B) Average size of the hydrodynamic diameter of the nanoherbicide. (C) Infrared spectroscopy. (D) Energy configuration between PLGA, atrazine and solvents.

Figure 3. Monte Carlo simulations. Panel A shows 20 different configurations of atrazine (in licorice and different colors) with a PLGA polymer (ball and stick model) from a Monte Carlo sampling. These configurations represent the lower energies from the single-point calculation performed with the Mopac PM7 method. Panel B shows the optimal best configuration of atrazine, which was calculated with Gaussian HF/3-21G. In this figure, we can observe a hydrogen bond formed between the hydrogen atom of atrazine and the oxygen atom of the ester group of PLGA. Because oxygen is an electronegative atom, it allows the formation of that type of bond. In panels A and B, hydrogen, oxygen, carbon, and nitrogen atoms are indicated in white, red, cyan, and blue, respectively.

seem to suggest that in a mixture of atrazine with acetonitrile and PLGA, the interaction between atrazine and acetonitrile will be more favorable than the interaction between atrazine with PLGA, thus reducing the amount of atrazine encapsulated in the PLGA matrix. Also, these results seem to explain our previous experimental findings in which we found that the atrazine encapsulation efficiency in the polymeric particles was 8% when acetonitrile was used to dissolve PLGA. Because PLGA and atrazine have almost an equal affinity for acetone, it is likely that the atrazine encapsulation efficiency in the

degrade the nanoherbicide at each time point during the atrazine release studies and thus to quantify the atrazine encapsulation efficiency in the polymeric particles. The energy interaction shows that atrazine has a better chemical affinity for acetonitrile (−4.5 kcal/mol) compared to the one that it has with acetone (−4 kcal/mol) and PLGA (−3.9 kcal/mol). On the other hand, acetone has almost the same energy interaction for PLGA (−3.7 kcal/mol) and atrazine (−4 kcal/mol), while acetonitrile has better energy interaction with atrazine (−4.5 kcal/mol) than with PLGA (−4.2 kcal/mol). These results E


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and therefore they might cause a harmful effect to the environment. Assessment of the Growth of the Plant Using an in Vitro Model. We conducted in vitro plant studies to assess the growth of the target species using in vitro plant models exposed to plant culture medium, PLGA nanoparticles, commercial atrazine, and PLGA nanoherbicides. The results show that for all the variables that we assessed in the in vitro plant studies such as stem length, root length, numbers of leaves, and fresh and dry weight, we observed a similar trend. The samples treated with atrazine and nanoherbicides underwent a significant decrement in the stem length, root length, fresh and dry weight, as well as, in the number of leaves. These results indicate the inhibition of growth due to atrazine (Figure 5). Furthermore, the presence of atrazine-free PLGA particles had a negligible effect on these variables in the target plants. As a matter of fact, the stem and root length of the target species treated with PLGA nanoparticles were comparable with that of the control sample in which the plants were only cultured with complete plant medium (Figure 5A,B). Also, we did not observe significant changes in the fresh weight of the plant or the number of leaves between the control and the atrazine-free PLGA nanoparticles (Figure 5C,D,E). Nevertheless, there is a statistical difference in the dry weight of plants treated with PLGA and control. Furthermore, there is a significant difference between the control and the effect produced by atrazine and nanoherbicides in the target species. As we increased the atrazine and nanoherbicides’ concentration in the target species, we observed a reduction in all variables. Among all the variables evaluated, it seems that the root length was the most affected by atrazine and nanoherbicides. It was found that free atrazine at the dose of 0.7 ug/mL and 6.6 ug/mL does not present significant differences with respect to the control, but when we encapsulated atrazine in the polymer matrix, we observed the lowest plant growth in comparison to the control for these doses. This means that the encapsulation of atrazine in the polymeric matrix enhances the effects of the plant roots regardless of the low doses of the active ingredient. We observed a significant toxic effect in the plants when the plants were treated with the highest dose of free atrazine and nanoherbicides. Moreover, these results confirm the atrazine release performance of nanoherbicides as the polymer matrix degrades, and therefore, atrazine is released in a controlled fashion. Thus, these results show the potential use of nanoherbicides in agriculture as it can be conceived as an alternative way to deliver the herbicide specifically in the target organism. As discussed previously, herbicides are used to kill weeds, which are plants that steal nutrients, light, and physical space from healthy plants. The invasion of weeds in crops significantly reduces the yield crop. These molecules of this group can be classified according to their mode of action because of the enzyme inhibition or prevention of cellular growth.31,32 In this study, we used atrazine as the active ingredient. Atrazine is a type of herbicide that interrupts photosynthesis once it reaches the chloroplast. More specifically, atrazine blocks the flow of electrons in photosynthesis II, inducing the inhibition of the assimilation of CO2 and the generation of large amounts of reactive oxygen species.33 Recently, scientists have reported the synthesis of nanoherbicides using different polymers such as PCL, chitosan, etc. achieving higher efficacy in killing weeds while reducing

polymeric matrix is higher when acetone is used as solvent to dissolve PLGA. This phenomenon matches very well with the atrazine encapsulation efficiency that was achieved (50%) when acetone was used as the solvent to dissolve PLGA. Therefore, improvement of the atrazine encapsulation efficiency in the PLGA matrix could be achieved by finding a solvent that has a strong chemical affinity with both atrazine and PLGA to overcome or reduce the competition that occurs between the solvent-PLGA and solvent-atrazine during the nanoherbicide synthesis. Furthermore, through Monte Carlo simulations, we learned that the type of chemical interaction that occurs between atrazine and PLGA (75:25) is in general van der Waals interactions. However, the simulations also show that the most stable interaction between PLGA and atrazine takes place when the hydrogen atom of atrazine and the oxygen atom of the ester bond of PLGA form a hydrogen bond whose length is close to 1.9 Å, as shown in Figure 3. Release Profile of PLGA Nanoherbicides. The PLGA nanoherbicides release 50% of atrazine in 72 h, whereas it takes 5 h to release completely free atrazine15 (Figure 4). The

Figure 4. Nanoherbicide atrazine release profile. Fifty percent of atrazine is released in 72 h.

nanoherbicides’ drug release profile depends strongly on the copolymer ratio. In this study, the nanoherbicides were synthesized with 75:25 copolymer ratio, and the rationale behind that was given in Characterization of PLGA Nanoherbicides section. Also, the degradation rate of nanoherbicides will depend on the particle’s size. According to the DLS studies, the prelyophilized nanoherbicides’ size is about 110 ± 10 nm, which was used to conduct the atrazine release studies. Particles with this size degrade faster than particles of a bigger size. For example, Grillo et al. showed that atrazine-loaded PCL particles release 50% of atrazine in 36 h.16 It is worth mentioning that the tunable degradation profile is a unique property of PLGA, not observed in other biodegradable polymers such as polyhydroxyburyrate (PHB) and Polyhydroxyvalerate (PHV). Furthermore, another great advantage of PLGA is that after the hydrolysis of nanoparticles, the remaining PLGA matrix is metabolized by the Kreb’s cycle as explained above, contrary to what is observed in particles that deliver the active ingredient only via the diffusion process. If the polymer is not biodegradable, those particles might still exist in the environment after they have released their payload, F


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Figure 5. Evaluation of the average stem (A) and root length (B) as well as the number of leaves (C) and fresh (D) and dry weight (E). Each letter indicates statistically significant difference. Welch’s ANOVA welch (p < 0.05) and Games Howell (p < 0.05) tests were performed.

toxicity in the environment and food. For example, Grillo et al. in 201216 and Pereira et al. in 20148 synthesized nanocapsules that transport atrazine using PCL, an aliphatic polyester, that improves the stability of the formulation. Pereira et al. assessed the effect of these nanoherbicides in beets (Brassica sp.) while growing corn (Zea mays).8 It was found that the germination index of corn was not affected by the nanoherbicides and biopolymer but that the nanoherbicide was able to control the beets, which confirms that the encapsulation process does not affect the chemical activity of the herbicide. These results coincide with our findings with PLGA nanoherbicides, where the encapsulation of atrazine in the polymeric PLGA particles

did not affect its herbicide activity. Also, the polymer itself did not generate toxic effects in plants. On the other hand, Oliveira et al. in 2015 published that atrazine encapsulated in PCL was 10 times more effective in inducing effects in growth, physiology, and oxidative stress than the commercial atrazine.32 It is known that the materials that are used in the synthesis of nanoherbicides can produce phytotoxicity effects in plants. In particular, they can cause changes in the germination index, root length, dry mass, as well as contribute to the germination and development of the plants.34 In this study, the use of the PLGA polymer did not affect the growth and development of the plant because in most cases, the stem and root length of PLGA was comparable G


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Figure 6. Cellular structure of the control and samples treated with three different nanoherbicides’ doses. (A) Control, (B) 0.7 μg/mL, (C) 6.3 μg/ mL, and (D) 54 μg/mL; n = nucleus, cw = cell wall, and v = vesicles. In A−C, we observe evidence that indicates that the plant cells is about to undergo cellular division because of the great number of vesicles (v) derived from diverse organelles and cytoplasm. It is important to note that in D, we can see cells with damage in the ultracellular structure. It is evident that the cell wall (cw) has lost rigidity. Also, we noticed irregularities of the plants’ nucleus (n), which coincides with evidence in Figure 5B in which there is significant difference for the 54.0 μg/mL of free and encapsulated atrazine dose in relation to the control. Also, at this dose, we observe the lowest stem’s growth. All the micrographics were obtained at 100×.

inhibiting growth as atrazine, even with 50% encapsulation efficiency. As the encapsulation efficiency is improved, the effectiveness of the nanoherbicide might increase. To observe the cellular alterations that the nanoherbicides might produce in the meristem of the plants’ root, we analyzed the plant cells exposed to different treatments. We conducted these studies using a light microscopy. Figure 6 shows in detail the cellular structure of the target species in each case. In Figure 6A−C, we showed evidence related to the target species’ cellular preparation for its next division because we can observe many vesicles originating in different organelles and cytoplasm.35 It is important to note that in Figure 6D we can observe a cellular detriment, which corresponds with the evidence in Figure 6B in which there is a significant difference in cellular response by the target species due to the 54.0 μg/ mL dose of atrazine and nanoherbicide compared with the control’s cellular structure. Also, at this dose, we can observe a decrement in the mean of the radical length of the plant. Pereira et al. determined the genotoxicity in the roots when they applied the nanocapsules of atrazine synthesized with PCL. These results indicated an interaction or possible absorption of the nanoparticles with the roots, which improved the released of the herbicide in the plant tissue. In this study, we observed toxicity in the plant cells through the assessed variables and structural analysis when we incremented the atrazine dose in both PLGA nanoherbicides and free atrazine (Figures 5 and 6). In conclusion, in this article, we report for the first time the synthesis of PLGA nanoherbicides with an encapsulation efficiency of 50% atrazine as active ingredient. The PLGA

to that of the control. Interestingly, in some cases, the PLGA nanoparticles behave as a growth potentiator in the observable variable like in the case of root length and the number of leaves (Figure 5B,C). Given the increased interactions among the nanoherbicide particle shell, and the root, we expected to see a large increase in the inhibitory effect of PLGA based-nanoherbicides over traditional atrazine. However, the PLGA nanoherbicides used in the in vitro experiments did not show significant improvements in reducing growth over the pure atrazine. As shown in Figure 5, the nanoherbicide treated plants had similar root length, fresh weight, dry weight, and number of leaves to those in the corresponding atrazine treatment. These results show that although the nanoherbicide was effective at inhibiting growth, it did not inhibit the growth of the plant more significantly than the pure atrazine. The reason for this result most likely is the 50% encapsulation efficiency. Although PLGA nanoherbicides’ encapsulation efficiency is not as high as other carriers such as PCL and chitosan in which a 90% EE was achieved, the efficacy of PLGA nanoherbicides may still provide benefits to agriculture. For example, nanoherbicides with small size are more likely to overcome the primary physiological barriers in the plant which often includes the root cortex, epidermis of the leaves, and stomata. In our case, the fact that lyophilized nanoherbicides have an average size of 500 nm ±10 nm is suitable to pass the potato plant because the pore-size of our biological model is 700 nm. Thus, small nanoparticles can effectively deliver the nanoherbicides in the target organism. Furthermore, the results clearly indicate that the nanoherbicide is at least as effective at H



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ABBREVIATIONS USED PLGA, poly(lactic-co-glycolic acid); DSPEPEG-NH2, 1,2distearoyl1-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]; DLS, dynamic light scattering; TEM, transmission electron microscopy; LC-MS, liquid chromatography/mass spectroscopy; diH2O, deionized water

polymer is an excellent option for the synthesis of novel nanoherbicides because of its biocompatibility, FDA approval, customized herbicide release, and tunable biodegradable profile. We would expect to achieve higher efficacy in inhibiting the growth as we increase the herbicides’ encapsulation efficiency. We found that lyophilized nanoherbicides whose size is around 500 nm ±10 nm affect plant growth. Since the nanoherbicides are made of the PLGA polymer, and this polymer degrades in the presence of water, we had to lyophilize the nanoherbicides to conduct the in vitro plant studies. The 50% encapsulation efficiency of atrazine in the PLGA matrix produced a decrement of 40% in root length. Also, we observed that the PLGA nanoherbicides are as effective as unencapsulated atrazine to reduce growth but substantially less toxic because after the PLGA matrix undergoes degradation, it will be metabolized by the Kreb’s cycle releasing only CO2 and water as final products. Therefore, PLGA nanoherbicides will not induce detrimental effects in the environment compared to other types of nanoherbicides or naked herbicides. Equally important, because PLGA nanoherbicides control the release of the active ingredient, they will greatly reduce toxicity in the environment. Notably, we observed that PLGA serves as a growth potentiator for plants. Thus, in this article we presented the proof-of-concept of using PLGA nanoherbicides as an alternative method for inhibiting weed growth and PLGA nanoparticles as a potential growth factor for plants. For atrazine-resistant crops such as corn, the PLGA nanoparticles could serve to promote crop growth while the encapsulated atrazine inhibits weed growth. Additionally, this growth effect could serve to counterbalance some of the effect of the herbicide on nontarget plants without atrazine resistance. Future studies will involve an in-depth understanding of the mechanism of plant−nanoherbicide interaction as well as the role of PLGA as a growth potentiator.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01911. TEM micrograph and average hydrodynamic diameter of lyophilized nanoherbicides (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 703-993-5895. E-mail: [email protected]. ORCID

Carolina Salvador-Morales: 0000-0002-2588-6608 Funding

This work was supported by a CSM startup fund (162904). This project was also funded in part through the Undergraduate Research Scholars Program sponsored by the George Mason Office of Student Scholarship Creativity and Research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Thomas Huff for assistance in developing the protocol for the LC-MS measurements. Furthermore, we thank Drs. Mikell Paige and Barney Bishop for giving access to the FT-IR and DLS instruments. I


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