Engineering Atrazine Loaded Poly (lactic-co-glycolic Acid)Nanoparticles to Ameliorate Environmental ChallengesBrian Schnoor,†,‡ Ahmad Elhendawy,†,‡ Suzanna Joseph,†,‡ Mark Putman,†,‡ Randall Chacon-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, Virginia20110, United States§InstitutoTecnologico 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
*S Supporting Information
ABSTRACT: The use of herbicides plays a vital role in controlling weeds and conserving crops; however, its usage generatesboth environmental and economic problems. For example, herbicides pose a financial issue as farmers must apply largequantities to protect crops due to absorption rates of less than 0.1%. Therefore, there is a great need for the development of newmethods 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 herbicidalactivity of the engineered PLGA nanoherbicides. Our method produced nanoherbicides with an average size of 110 ± 10 nmprior to lyophilization. Fifty percent of the loaded atrazine in the PLGA matrix is released in 72 h. Furthermore, we performedMonte Carlo simulations to determine the chemical interaction among atrazine, PLGA, and the solvent system. One of the mostsignificant 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 PLGAnanoparticles, potato plants undergo a significant decrease in stem length, root length, fresh weight, dry weight, and the numberof leaves, with root length being the most affected. These experimental results suggest the herbicidal effectiveness of atrazine-loaded PLGA nanoherbicides in inhibiting the growth of the potato plant. Hence, we present the proof-of-concept for usingPLGA nanoherbicides as an alternative method for inhibiting weed growth. Future studies will involve a deep understanding ofthe mechanism of plant−nanoherbicide interaction as well as the role of PLGA as a growth potentiator.
■ INTRODUCTIONProtecting crops from nutrient stealing weeds is an essentialpart of agriculture. Herbicides play a vital role in controllingweeds and conserving crops. However, the use of herbicidesgenerates many environmental and economic problems.Current herbicides pose a significant risk to the environmentsince vast quantities of herbicides are washed into streams andrivers as runoff, which can kill nontarget organisms and disruptecosystems.1−4 The widespread use of herbicides, such asatrazine, also presents economic problems because theseherbicides evaporate quickly and are readily trapped in thetop layer of soil due to soil absorption.5,6 Therefore, less than0.1% of the applied herbicides reach the target organisms.7
Even the herbicide that reaches the target weed can beineffective due to poor translocation in the weed and thedevelopment of herbicide resistant weeds. Because of thisphenomenon, larger amounts of herbicides are required, whichcan exacerbate environmental damage. To overcome theseproblems, a new delivery system is needed to protect theherbicides, ensure that they reach the weed, and improvetransport of the herbicide within the target weed.
Atrazine is the second most popular herbicide in the UnitedStates since it is very efficient in controlling weeds and hasproven 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 thanany other pesticide.9 In the European Union, atrazine hasalready been banned because atrazine contamination in thegroundwater exceeded the maximum limits set by law.10
Furthermore, studies indicated that atrazine has harmful effectson nontarget organisms in aquatic ecosystems.3,4,11
Nanoparticle-based delivery systems for herbicides alsoknown as “nanoherbicides” have shown great promise toimprove herbicidal efficacy.8,12−14 Nanoherbicides consist of atraditional 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, 2018Revised: June 14, 2018Accepted: July 13, 2018Published: July 24, 2018
Article
pubs.acs.org/JAFCCite This: J. Agric. Food Chem. 2018, 66, 7889−7898
evaporation of naked herbicides and improve both theabsorption of herbicides through the plant root and trans-location within the plant.8,12−14 Furthermore, nanoherbicidescan reduce the amount of herbicide trapped in the top layer ofsoil, consequently diminishing water contamination due torunoff removed from surface soil. Similarly, PEGylation of thenanoparticles’ surface can also improve absorption andtranslocation of the nanoparticles in the plant and decreasethe amount of free herbicide that could contaminate streamsand reach nontarget organisms. Thus, we can reduce theamount of herbicide delivered to nontarget species insurrounding or downstream areas by increasing the efficiencyof delivering the herbicide to the target weed.Several research groups have synthesized a wide variety of
nanoherbicides that combine surfactants, polymers, andmetallic nanoparticles to facilitate the application of herbicidesin crop fields by overcoming the insolubility of someherbicides. 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 increasethe solubility of poorly water-soluble active ingredients (AI).14
There are other types of polymer-based nanoherbicides thatseek to release the payload in a slow, sustained fashion whilepreventing premature degradation of the active ingredient.This class of nanoherbicides includes polymers such aspoly(epsilon-caprolactone) (PCL),8,15,16 chitosan,17 and algi-nate.18
Here, we report a novel nanoherbicide delivery system basedon a PLGA polymer. PLGA is a biocompatible, biodegradable,and Food and Drug Administration (FDA) approvedcopolymer formed by two different monomers: lactide andglycolide. PLGA provides several significant advantages overother polymer-based nanoherbicides. First, the degradationrate of this copolymer can be manipulated according to ouragricultural needs by varying the ratio between lactide andglycolide monomers. The degradation of PLGA occurs viahydrolysis, which cleaves the ester bonds of the lactide andglycolide monomers. The ester bonds of the lactide monomersundergo a much slower degradation rate due to the steric effectof the CH3 group that is present in the lactide but not in theglycolide monomer.19 This chemical feature allows the tuningof the degradation profile of the PLGA nanoherbicide, whichcannot be achieved with the PCL homopolymer. Moreover,chitosan, a highly hydrophilic polymer17 cannot be used tosuccessfully encapsulate atrazine. Furthermore, PLGA has adegradation-based release profile (i.e., the active ingredient isreleased in unison with the degradation of the nanoparticle).This will prevent the problem of rapid separation of the activeingredient from the nanocarrier after reaching the soil/plantsurface, which can significantly affect the transport of the activeingredient.20−22 Indeed, PCL also follows this degradation-based release profile and achieves a similar release effect.However, PCL presents its own environmental challenge dueto its slow degradation rate. Unlike PLGA, which willcompletely degrade within 6 months at the most, PCLdegrades in 1−3 years.23 This is an extended period of time forPCL to interact with organisms in the environment. Addition-ally, other polymers such as alginate are nontoxic only when inhigh purity. Obtaining that level of purity often involvesextensive and expensive synthesis processes.24
PLGA, however, is a harmless polymer that degradescompletely into nontoxic byproducts. In fact, when hydrolysistakes place, the lactide and glycolide monomers are trans-
formed into lactic and glycolic acid, respectively, which aremetabolized by the Kreb’s cycle. The byproducts of the Kreb’scycle are carbon dioxide and water. Therefore, PLGA is nottoxic to humans, plants, or animals.19 Additionally, it is worthmentioning that PLGA can currently be purchased inkilograms or tons at low cost from Chinese manufacturers.Thus, the scaled-up production of PLGA nanoherbicides maynot represent a technical challenge for the use of PLGAnanoherbicides in agricultural settings.
■ MATERIALS AND METHODSMaterials. The reagents atrazine, terbuthylazine, acetone,
acetonitrile, formic acid, and ethanol were purchased from Sigma-Aldrich. Lecithin was acquired from Alfa Aesar, and PLGA wasobtained from Lactel Absorbable Polymers. 1,2-Distearoyl-sn-glycero3-phosphoethanolamine-N-[amino(polyethylene glycol)](DSPE-PEG-NH2) was purchased from Laysan Bio Inc. Uranylacetate and the TEM copper mesh grids were acquired from ElectronMicroscopy Sciences.
Synthesis of PLGA Nanoherbicides. PLGA nanoherbicideswere synthesized using a modified nanoprecipitation methoddeveloped in-house. Briefly, we prepared stock solutions of 1 mg/mL DSPE-PEG-NH2 and 1 mg/mL Lecithin in 4% ethanol. 900 μL ofthe DSPE-PEG-NH2 solution and 1200 μL of the lecithin solutionwere 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 to68 °C for 5 min under light stirring using an IKA hot plate. A stocksolution of 2 mg/mL of PLGA in acetone was prepared.Subsequently, 1 mL of the PLGA solution and 2 mg of atrazinewere added to a smaller glass vial. Using a 25 G 1 × 1.5 needle andplastic syringe, the organic phase was added dropwise to the aqueousphase under moderate stirring. The needle was submerged intosolution and slowly moved up and down as the organic phase wasadded to the aqueous phase to help prevent particle aggregation. Tosynthesize larger quantities of nanoherbicides, this procedure wasscaled up 5-fold. To accomplish this task, the same stock solutionswere used to prepare an aqueous solution consisting of 60 mL ofdiH2O, 4.5 mL of DSPE-PEG-NH2 stock solution, and 6 mL oflecithin stock solution. This aqueous phase was then heated to 68 °Cfor 8 min under light stirring. Then, 5 mL of PLGA stock solution wasadded to a glass vial containing 10 mg of atrazine. The organicsolution was then added to the aqueous solution using the samemethod described above.
Characterization of PLGA Nanoherbicides. Particle Size ofPLGA Nanoherbicides. The nanoherbicide size was determined bythe dynamic light scattering (DLS) technique. The DLS measure-ments were taken with an N5 Submicron Particle Size Analyzer(Beckman Coulter). DLS was first calibrated using standardizedpolymer beads of 100 nm in size. Then, 3 μL of the nanoherbicidesolution was mixed with 2 mL of diH2O in a plastic cuvette. Thecuvette was then placed in the DLS for analysis at 25 °C and 90°deflection.
Morphology of PLGA Nanoherbicides. The morphology of PLGAnanoherbicides was examined by a transmission electron microscope(TEM). These samples were prepared by pipetting 10 μL of thenanoherbicide sample onto the carbon side of a copper grid. Then 10μL of uranyl acetate was added to the copper grid, which was thenallowed to dry overnight. Next, the samples were examined with theTecnai G spirit BioTwin at 80 kV. Images were recorded with anAMT 2k CCD camera.
Fourier Transform-Infrared (FT-IR) Spectroscopy Character-ization. The PLGA nanoherbicides were characterized with a JascoFT/IR 4100 spectrophotometer, operated in the range 550−4000cm−1 employing 36 scans per sample and a resolution of 4 cm−1. TheFT-IR measurements were performed on nanoherbicides (i.e.,atrazine-loaded PLGA nanoparticles), unloaded PLGA nanoparticles(i.e., PLGA polymeric particles without atrazine) and atrazine. Foreach FT-IR spectrum, a sample of 2 mg was mixed with 200 mg ofsodium bromide using a mortar and pestle before being compressed
Journal of Agricultural and Food Chemistry Article
into a flat translucent disk. Subsequently, each sample was analyzedusing an FT/IR spectrophotometer, and peaks were identified.Encapsulation Efficiency of PLGA Nanoherbicides. The
percentage of atrazine encapsulated in the PLGA polymer matrixwas determined using a liquid chromatography/mass spectrometry(LC-MS) technique. After nanoherbicide synthesis, we degraded thenanoherbicides with acetonitrile. One milliliter of acetonitrile wasadded to the nanoherbicide solution until it was perfectly clear. Next,the resulting solution was vortexed for 3 min. Subsequently, thesolution was centrifuged in an Amicon filter with 100 kDa molecularcutoff at 2000 rpm, allowing the atrazine to pass into the filtrate. Thefiltrate was then protected from light exposure to prevent atrazinedegradation. 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 concentrationusing the LC-MS technique. To accomplish this task, we prepared anatrazine stock solution of 1 mg/mL atrazine in methanol. Then, weprepared a stock solution of terbuthylazine (1 mg/mL) in acetone. Anacetonitrile mobile phase was prepared by mixing 99 mL of highperformance liquid chromatography (HPLC) grade acetonitrile and 1mL of formic acid to produce 1% formic acid in acetonitrile. A diH2Omobile phase was also prepared by mixing 1 mL of formic acid and 99mL of diH2O. Then, the complete mobile phase was prepared bymixing 50 mL of acetonitrile mobile phase and 50 mL of diH2Omobile phase. Standard 1 was prepared by adding 75 μL ofterbuthylazine stock solution, 7.5 μL of atrazine stock solution, and1417.5 μL of complete mobile phase to an HPLC vial to produce a 5ng/μL atrazine concentration. Standard 2 was prepared using 75 μLof terbuthylazine stock solution, 22.5 μL of atrazine stock solution,and 1402.5 μL of mobile phase to obtain an atrazine concentration of15 ng/μL. Standard 3 was prepared using 75 μL of terbuthylazinestock solution, 50 μL of atrazine stock solution, and 1375 μL ofmobile 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 toproduce an atrazine concentration of 45 ng/μL. Finally, standard 5was prepared using 75 μL of terbuthylazine stock solution, 100 μL ofatrazine stock solution, and 1325 μL of mobile phase to obtain a66.67 ng/μL atrazine concentration. These five internal standardswere diluted 1000-fold in acetonitrile and analyzed with LC-MS using1% formic acid in acetonitrile mobile solvent. The internal standardsare 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 μLof the terbuthylazine internal standard prepared above and 1125 μL ofcomplete mobile phase. This mixture resulted in a 5-fold dilution ofthe degraded nanoherbicide sample. The samples were further diluted1000-fold in acetonitrile. Then, the diluted solution was analyzedusing the LC-MS instrument and measured against the calibrationcurve created from the standard samples. We determined theconcentration of atrazine in each nanoherbicide sample based onthis calibration curve.Molecular Interaction Characterization among PLGA,
Atrazine, and Solvents. We performed a configurational samplingusing a Monte Carlo strategy to study the chemical interaction amongPLGA, atrazine, acetonitrile, and acetone as implemented in Avila etal.25 The energy interaction analysis between the pairs of PLGA andthe various molecules (e.g., atrazine, acetonitrile, and acetone) wasperformed using a total of 200,000 pair configurations. The energyinteraction for each pair configuration was computed using MOPAC2016 and the semiempiric PM7 method.26,27 The atrazine,acetonitrile, and acetone molecules were geometrically optimizedusing 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 dialysisalong with the LC-MS technique to determine the percent of theencapsulated atrazine released over time. According to the nano-herbicide synthesis described above, 100 μL of the nanoherbicidesolution was added to 6 mini-dialysis tubes with a pore size of 3600Da. Then, these dialysis tubes were placed in a 2 L reservoir of
deionized water and stirred with a magnetic stirrer. Then, theminidialysis tubes were removed from the dialysis reservoir at 6, 12,24, 36, 48, and 72 h time points. The volume contained in the mini-dialysis tubes at each time point was transferred to a glass vial, and anequal volume of acetonitrile was then added and vortexed to degradethe nanoparticles. All sample solutions were then diluted to 1050 μLusing acetonitrile. This was repeated 3 times to obtain triplicatesamples for each time point. The concentration of atrazine in each ofthese samples was then determined using an LC-MS instrument.
Lyophilization Process of PLGA Nanoherbicides. Thelyophilization process plays a key role in nanoherbicide synthesis toprevent PLGA degradation and facilitate the storage of nano-herbicides until their use. Several cryoprotectants, including sucroseand various polymers at different concentrations, were tested. Fivepercent of sucrose was the best cryoprotectant found because itgreatly prevented the enlargement of the nanoparticle size. Thus, 5%sucrose was mixed with the filtered nanoherbicide solution in a 1 to 1ratio before the solution was lyophilized. After lyophilization, thenanoherbicides were then resuspended to test the nanoherbicides’solubility.
In Vitro Plant Studies to Assess the Herbicide Activity ofNanoherbicides. We used potato plants as a biological model toassess the herbicidal activity of the engineered nanoherbicides(Biotechnology Research Center, Instituto Tecnologico de CostaRica, Cartago, Costa Rica). The in vitro plants were reproduced incomplete culture medium following the protocol described in Floreset al.29 The plants were inoculated in microcuttings, three for eachcontainer. Twenty milliliters of complete culture medium was usedper container. They were incubated at 22 °C in a controlledtemperature environment with a photoperiod of 16/8 h. for a periodof 22−30 days.
Assessment of the Growth and Development of the TargetSpecies Using an in Vitro Model. We assessed in vitro the effect offree herbicide and encapsulated herbicide in the PLGA matrix inplants with respect to the growth and development of the targetorganism (i.e., potato plant) using different atrazine concentrations.We used atrazine with 98% purity, and the nanoherbicides werecomposed of 42.38% atrazine and 57.62% PLGA, and lyophilized with5% sucrose. Unloaded PLGA nanoparticles and the targeted organismcultured only with complete medium were the control experiments ofthis study. The concentrations of PLGA nanoparticles, atrazine, andnanoherbicides used in this study are shown in Table 1. The
concentrations of nanoherbicide used were selected to account for themass of the nanoparticle shell and administer the same amount ofatrazine as the pure atrazine samples. Similarly, the concentrations ofPLGA particles without atrazine were selected to administer the sameamount of PLGA as the nanoherbicide trials. In the in vitro studies,we evaluated the following key variables: stem length, longest rootlength, number of leaves, and fresh and dry weight. We conductedthese experiments in 50 replicates per treatment.
Table 1. Targeted Organism (Potato Plant) Treated withComplete Medium, Atrazine, Nanoherbicides, and PLGANanoparticles at Different Concentrations
Determination of Altered Cellular Division. We selected theroot 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 partof the root tip to analyze with the light microscope to investigate if thetreatment produced morphological cellular alterations. We labeledthis sample with Giemsa (3:1). We placed the samples on amicroscope slide, and we added a drop of the dye. Then, we placed acoverslip 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 withinthe 95% confident interval, p = 0.05, using the statistical programMinitab 17.30
■ RESULTS AND DISCUSSIONPLGA Nanoherbicide’s Design. In the core−shell
structure of the nanoherbicide, PLGA forms the core, andthe shell is made of DSPE-PEG-NH2. Herbicides such asatrazine are embedded in the PLGA polymer matrix (Figure1).
Characterization of PLGA Nanoherbicides. Nano-herbicides have been synthesized with different polymerssuch as PCL and chitosan. To the best of our knowledgePLGA nanoherbicides have not been synthesized yet. Thus, weare the first research group reporting their synthesis,characterization, and herbicidal activity. The use of PLGA inagricultural settings has not been yet explored. We realized thegreat benefits that this polymer could have offered toagriculture because of its unique physicochemical properties.For example, since PLGA is a copolymer, we can fine-tune thedegradation characteristics of PLGA nanoherbicides by varyingthe copolymer ratio. This specific functionality cannot becompletely achieved by other polymers such as PCL andchitosan because they are not copolymers. Also, the fact thatPLGA is a copolymer allows it to encapsulate nonwater-solubleherbicides such as atrazine. In this study, we used a 75:25copolymer ratio, 75% of lactide, and 25% of glycolidemonomers because this copolymer ratio makes the entirePLGA matrix more hydrophobic due to the steric effects givenby the methyl group present in the lactide monomer. Atrazinecan then be encapsulated in the polymer matrix. Additionally,the ratio in PLGA (75:25) still maintains enough hydrophilic
glycolide monomers to release the herbicide in a controlledrelease fashion over a relatively short period of time.
Transmission Electron Microscopy and DynamicLight Scattering. TEM revealed that PLGA nanoherbicideshave a very well-defined spherical shape (Figure 2A). DLSresults show that the average size of nanoherbicides in diH2Ois 110 ± 10 nm before lyophilization (Figure 2B). Thesenanoherbicides remained in suspension for several weeks aftertheir synthesis. We lyophilized the nanoherbicides after theirsynthesis to conduct in vitro plant studies. Before thelyophilization process, we incorporated 5% sucrose in thenanoherbicide solution to avoid precipitation after the freeze-drying step. However, we observed a substantial increment inthe nanoherbicide size after the lyophilization process. The sizeof the freeze-dried nanoherbicides is about 500 nm ±10 nm(Figures S1,S2). Subsequently, we tested the stability of thesenanoherbicides in diH2O after the lyophilization process, andwe found that they remained in suspension for several weeks.
Molecular Interaction Characterization among PLGA,Atrazine, and Solvents. The encapsulation efficiency for a 5-fold scaled-up prelyophilized nanoherbicide sample is 50%.Infrared studies demonstrated the association between atrazineand the polymeric chains of PLGA as shown in Figure 2C. TheIR of atrazine shows a band at 3258 cm−1 which correspondsto stretching of the N−H bond present in the amine functionalgroup of atrazine, whereas the band at 2973 cm−1 is associatedwith the stretching of the alkyl group C−H bond. The bands at1622 and 1557 cm−1 indicated the deformation of CC andCN bonds, respectively, in atrazine. PLGA polymericnanoparticles (e.g., unloaded nanoherbicides) showed bandsat 3388 cm−1 (O−H bond stretching), 1759 cm−1 (stretchingof the carbonyl CO bond), and 1068 cm−1 (angulardeformation of C−O). The IR spectrum of PLGA nano-herbicides (e.g., atrazine-loaded PLGA nanoparticles) showedthree distinctive peaks at 3389 cm−1, 1760 cm−1, and 1069 cm1
that correspond to the PLGA component of the nanoherbicidesample, while the peaks at 1623 and 1555 cm−1 correspond toatrazine’s bands. Thus, these peaks indicate the presence ofatrazine in the polymeric matrix of the nanoherbicide.Grillo et al. achieved between 80% to 90% atrazine
encapsulation efficiency using an interfacial deposition ofpreformed polymer method and PCL as a polymer matrix.16
This process produced atrazine-loaded nanoherbicides ofapproximately 200−300 nm size before lyophilization.Although our modified nanoprecipitation method providesatrazine-loaded PLGA nanoparticles with lower encapsulationefficiency, the particle size of our prelyophilized nanoherbicidesample is 110 ± 10 nm. Pereira et al. reported theencapsulation efficiency of PCL nanocapsules and nanospheresof 93%.8 The size of the nanocapsules and nanospheres was513 ± 7.5 nm and 365.1 ± 0.16 nm, respectively. Thus, itseems that the encapsulation efficiency is related to theparticle’s size. The higher the particle size, the higher is theencapsulation efficiency.To further investigate the parameters that affect the
nanoherbicides’ encapsulation efficiency, we performedquantum mechanics calculations to determine the type ofinteraction energy that takes place among PLGA, atrazine, andsolvents (e.g., acetonitrile and acetone) as well as themagnitude of such interaction (Figure 2D). It is importantto mention that we used acetone to dissolve PLGA during thenanoprecipitation method as described in the PLGA nano-herbicides’ synthesis. We only used acetonitrile as a solvent to
Figure 1. Nanoherbicide’s design. Nanoherbicides are formed by aself-assembly process using a modified nanoprecipitation methoddeveloped in-house. The nanoherbicide is composed of, lecithin, lipid-PEG-NH2 and PLGA. Atrazine is encapsulated in the polymericmatrix.
Journal of Agricultural and Food Chemistry Article
degrade the nanoherbicide at each time point during theatrazine release studies and thus to quantify the atrazineencapsulation efficiency in the polymeric particles. The energyinteraction shows that atrazine has a better chemical affinity foracetonitrile (−4.5 kcal/mol) compared to the one that it haswith acetone (−4 kcal/mol) and PLGA (−3.9 kcal/mol). Onthe other hand, acetone has almost the same energy interactionfor PLGA (−3.7 kcal/mol) and atrazine (−4 kcal/mol), whileacetonitrile has better energy interaction with atrazine (−4.5kcal/mol) than with PLGA (−4.2 kcal/mol). These results
seem to suggest that in a mixture of atrazine with acetonitrileand PLGA, the interaction between atrazine and acetonitrilewill be more favorable than the interaction between atrazinewith PLGA, thus reducing the amount of atrazine encapsulatedin the PLGA matrix. Also, these results seem to explain ourprevious experimental findings in which we found that theatrazine encapsulation efficiency in the polymeric particles was8% when acetonitrile was used to dissolve PLGA. BecausePLGA and atrazine have almost an equal affinity for acetone, itis likely that the atrazine encapsulation efficiency in the
Figure 2. Characterization of PLGA nanoherbicides. (A) TEM image of nanoherbicides’ morphology. (B) Average size of the hydrodynamicdiameter 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 performedwith the Mopac PM7 method. Panel B shows the optimal best configuration of atrazine, which was calculated with Gaussian HF/3-21G. In thisfigure, we can observe a hydrogen bond formed between the hydrogen atom of atrazine and the oxygen atom of the ester group of PLGA. Becauseoxygen is an electronegative atom, it allows the formation of that type of bond. In panels A and B, hydrogen, oxygen, carbon, and nitrogen atomsare indicated in white, red, cyan, and blue, respectively.
Journal of Agricultural and Food Chemistry Article
polymeric matrix is higher when acetone is used as solvent todissolve PLGA. This phenomenon matches very well with theatrazine encapsulation efficiency that was achieved (50%)when acetone was used as the solvent to dissolve PLGA.Therefore, improvement of the atrazine encapsulationefficiency in the PLGA matrix could be achieved by finding asolvent that has a strong chemical affinity with both atrazineand PLGA to overcome or reduce the competition that occursbetween the solvent-PLGA and solvent-atrazine during thenanoherbicide synthesis. Furthermore, through Monte Carlosimulations, we learned that the type of chemical interactionthat occurs between atrazine and PLGA (75:25) is in generalvan der Waals interactions. However, the simulations alsoshow that the most stable interaction between PLGA andatrazine takes place when the hydrogen atom of atrazine andthe oxygen atom of the ester bond of PLGA form a hydrogenbond 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 takes5 h to release completely free atrazine15 (Figure 4). The
nanoherbicides’ drug release profile depends strongly on thecopolymer ratio. In this study, the nanoherbicides weresynthesized with 75:25 copolymer ratio, and the rationalebehind that was given in Characterization of PLGA Nano-herbicides section. Also, the degradation rate of nanoherbicideswill depend on the particle’s size. According to the DLSstudies, 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 biggersize. For example, Grillo et al. showed that atrazine-loadedPCL particles release 50% of atrazine in 36 h.16
It is worth mentioning that the tunable degradation profile isa unique property of PLGA, not observed in otherbiodegradable polymers such as polyhydroxyburyrate (PHB)and Polyhydroxyvalerate (PHV). Furthermore, another greatadvantage of PLGA is that after the hydrolysis of nanoparticles,the remaining PLGA matrix is metabolized by the Kreb’s cycleas explained above, contrary to what is observed in particlesthat deliver the active ingredient only via the diffusion process.If the polymer is not biodegradable, those particles might stillexist in the environment after they have released their payload,
and therefore they might cause a harmful effect to theenvironment.
Assessment of the Growth of the Plant Using an inVitro Model.We conducted in vitro plant studies to assess thegrowth of the target species using in vitro plant modelsexposed to plant culture medium, PLGA nanoparticles,commercial atrazine, and PLGA nanoherbicides. The resultsshow that for all the variables that we assessed in the in vitroplant studies such as stem length, root length, numbers ofleaves, and fresh and dry weight, we observed a similar trend.The samples treated with atrazine and nanoherbicidesunderwent a significant decrement in the stem length, rootlength, 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 PLGAparticles had a negligible effect on these variables in the targetplants. As a matter of fact, the stem and root length of thetarget species treated with PLGA nanoparticles werecomparable with that of the control sample in which theplants were only cultured with complete plant medium (Figure5A,B). Also, we did not observe significant changes in the freshweight of the plant or the number of leaves between thecontrol and the atrazine-free PLGA nanoparticles (Figure5C,D,E). Nevertheless, there is a statistical difference in the dryweight of plants treated with PLGA and control. Furthermore,there is a significant difference between the control and theeffect produced by atrazine and nanoherbicides in the targetspecies. As we increased the atrazine and nanoherbicides’concentration in the target species, we observed a reduction inall variables. Among all the variables evaluated, it seems thatthe root length was the most affected by atrazine andnanoherbicides. It was found that free atrazine at the dose of0.7 ug/mL and 6.6 ug/mL does not present significantdifferences with respect to the control, but when weencapsulated atrazine in the polymer matrix, we observed thelowest plant growth in comparison to the control for thesedoses. This means that the encapsulation of atrazine in thepolymeric matrix enhances the effects of the plant rootsregardless of the low doses of the active ingredient. Weobserved a significant toxic effect in the plants when the plantswere treated with the highest dose of free atrazine andnanoherbicides.Moreover, these results confirm the atrazine release
performance of nanoherbicides as the polymer matrixdegrades, and therefore, atrazine is released in a controlledfashion. Thus, these results show the potential use ofnanoherbicides in agriculture as it can be conceived as analternative way to deliver the herbicide specifically in the targetorganism. As discussed previously, herbicides are used to killweeds, which are plants that steal nutrients, light, and physicalspace from healthy plants. The invasion of weeds in cropssignificantly reduces the yield crop. These molecules of thisgroup can be classified according to their mode of actionbecause of the enzyme inhibition or prevention of cellulargrowth.31,32 In this study, we used atrazine as the activeingredient. Atrazine is a type of herbicide that interruptsphotosynthesis once it reaches the chloroplast. Morespecifically, atrazine blocks the flow of electrons in photosyn-thesis II, inducing the inhibition of the assimilation of CO2 andthe generation of large amounts of reactive oxygen species.33
Recently, scientists have reported the synthesis of nano-herbicides using different polymers such as PCL, chitosan, etc.achieving higher efficacy in killing weeds while reducing
Figure 4. Nanoherbicide atrazine release profile. Fifty percent ofatrazine is released in 72 h.
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toxicity in the environment and food. For example, Grillo et al.in 201216 and Pereira et al. in 20148 synthesized nanocapsulesthat transport atrazine using PCL, an aliphatic polyester, thatimproves the stability of the formulation. Pereira et al. assessedthe effect of these nanoherbicides in beets (Brassica sp.) whilegrowing corn (Zea mays).8 It was found that the germinationindex of corn was not affected by the nanoherbicides andbiopolymer but that the nanoherbicide was able to control thebeets, which confirms that the encapsulation process does notaffect the chemical activity of the herbicide. These resultscoincide with our findings with PLGA nanoherbicides, wherethe encapsulation of atrazine in the polymeric PLGA particles
did not affect its herbicide activity. Also, the polymer itself didnot 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 ininducing effects in growth, physiology, and oxidative stressthan the commercial atrazine.32 It is known that the materialsthat are used in the synthesis of nanoherbicides can producephytotoxicity effects in plants. In particular, they can causechanges in the germination index, root length, dry mass, as wellas contribute to the germination and development of theplants.34 In this study, the use of the PLGA polymer did notaffect the growth and development of the plant because inmost cases, the stem and root length of PLGA was comparable
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 letterindicates statistically significant difference. Welch’s ANOVA welch (p < 0.05) and Games Howell (p < 0.05) tests were performed.
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to that of the control. Interestingly, in some cases, the PLGAnanoparticles behave as a growth potentiator in the observablevariable 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 increasein the inhibitory effect of PLGA based-nanoherbicides overtraditional atrazine. However, the PLGA nanoherbicides usedin the in vitro experiments did not show significantimprovements in reducing growth over the pure atrazine. Asshown in Figure 5, the nanoherbicide treated plants had similarroot length, fresh weight, dry weight, and number of leaves tothose in the corresponding atrazine treatment. These resultsshow that although the nanoherbicide was effective atinhibiting growth, it did not inhibit the growth of the plantmore significantly than the pure atrazine. The reason for thisresult 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 whicha 90% EE was achieved, the efficacy of PLGA nanoherbicidesmay still provide benefits to agriculture. For example,nanoherbicides with small size are more likely to overcomethe primary physiological barriers in the plant which oftenincludes the root cortex, epidermis of the leaves, and stomata.In our case, the fact that lyophilized nanoherbicides have anaverage size of 500 nm ±10 nm is suitable to pass the potatoplant because the pore-size of our biological model is 700 nm.Thus, small nanoparticles can effectively deliver the nano-herbicides in the target organism. Furthermore, the resultsclearly indicate that the nanoherbicide is at least as effective at
inhibiting growth as atrazine, even with 50% encapsulationefficiency. As the encapsulation efficiency is improved, theeffectiveness of the nanoherbicide might increase.To observe the cellular alterations that the nanoherbicides
might produce in the meristem of the plants’ root, we analyzedthe plant cells exposed to different treatments. We conductedthese studies using a light microscopy. Figure 6 shows in detailthe cellular structure of the target species in each case. InFigure 6A−C, we showed evidence related to the targetspecies’ cellular preparation for its next division because we canobserve many vesicles originating in different organelles andcytoplasm.35 It is important to note that in Figure 6D we canobserve a cellular detriment, which corresponds with theevidence in Figure 6B in which there is a significant differencein cellular response by the target species due to the 54.0 μg/mL dose of atrazine and nanoherbicide compared with thecontrol’s cellular structure. Also, at this dose, we can observe adecrement in the mean of the radical length of the plant.Pereira et al. determined the genotoxicity in the roots whenthey applied the nanocapsules of atrazine synthesized withPCL. These results indicated an interaction or possibleabsorption of the nanoparticles with the roots, which improvedthe released of the herbicide in the plant tissue. In this study,we observed toxicity in the plant cells through the assessedvariables and structural analysis when we incremented theatrazine 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 encapsulationefficiency of 50% atrazine as active ingredient. The PLGA
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 toundergo cellular division because of the great number of vesicles (v) derived from diverse organelles and cytoplasm. It is important to note that inD, 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 ofthe plants’ nucleus (n), which coincides with evidence in Figure 5B in which there is significant difference for the 54.0 μg/mL of free andencapsulated atrazine dose in relation to the control. Also, at this dose, we observe the lowest stem’s growth. All the micrographics were obtained at100×.
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polymer is an excellent option for the synthesis of novelnanoherbicides because of its biocompatibility, FDA approval,customized herbicide release, and tunable biodegradableprofile. We would expect to achieve higher efficacy ininhibiting the growth as we increase the herbicides’encapsulation efficiency. We found that lyophilized nano-herbicides whose size is around 500 nm ±10 nm affect plantgrowth. Since the nanoherbicides are made of the PLGApolymer, and this polymer degrades in the presence of water,we had to lyophilize the nanoherbicides to conduct the in vitroplant studies. The 50% encapsulation efficiency of atrazine inthe PLGA matrix produced a decrement of 40% in root length.Also, we observed that the PLGA nanoherbicides are aseffective as unencapsulated atrazine to reduce growth butsubstantially less toxic because after the PLGA matrixundergoes degradation, it will be metabolized by the Kreb’scycle releasing only CO2 and water as final products.Therefore, PLGA nanoherbicides will not induce detrimentaleffects in the environment compared to other types ofnanoherbicides or naked herbicides. Equally important,because PLGA nanoherbicides control the release of the activeingredient, they will greatly reduce toxicity in the environment.Notably, we observed that PLGA serves as a growthpotentiator for plants. Thus, in this article we presented theproof-of-concept of using PLGA nanoherbicides as analternative method for inhibiting weed growth and PLGAnanoparticles as a potential growth factor for plants. Foratrazine-resistant crops such as corn, the PLGA nanoparticlescould serve to promote crop growth while the encapsulatedatrazine inhibits weed growth. Additionally, this growth effectcould serve to counterbalance some of the effect of theherbicide on nontarget plants without atrazine resistance.Future studies will involve an in-depth understanding of themechanism of plant−nanoherbicide interaction as well as therole of PLGA as a growth potentiator.
■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jafc.8b01911.
TEM micrograph and average hydrodynamic diameterof lyophilized nanoherbicides (PDF)
■ AUTHOR INFORMATIONCorresponding Author*Tel: 703-993-5895. E-mail: [email protected] Salvador-Morales: 0000-0002-2588-6608FundingThis work was supported by a CSM startup fund (162904).This project was also funded in part through the Under-graduate Research Scholars Program sponsored by the GeorgeMason Office of Student Scholarship Creativity and Research.NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSWe thank Dr. Thomas Huff for assistance in developing theprotocol for the LC-MS measurements. Furthermore, we thankDrs. Mikell Paige and Barney Bishop for giving access to theFT-IR and DLS instruments.
■ ABBREVIATIONS USED
PLGA, poly(lactic-co-glycolic acid); DSPEPEG-NH2, 1,2-distearoyl1-sn-glycero-3-phosphoethanolamine-N-[amino-(polyethylene glycol)]; DLS, dynamic light scattering; TEM,transmission electron microscopy; LC-MS, liquid chromatog-raphy/mass spectroscopy; diH2O, deionized water
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