Positive-Charge Functionalized Mesoporous Silica Nanoparticles as

Article pubs.acs.org/JAFC

Positive-Charge Functionalized Mesoporous Silica Nanoparticles as Nanocarriers for Controlled 2,4-Dichlorophenoxy Acetic Acid Sodium Salt Release Lidong Cao,†,∥ Zhaolu Zhou,†,∥ Shujun Niu,‡ Chong Cao,† Xiuhuan Li,† Yongpan Shan,† and Qiliang Huang*,†

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†

Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, P. R. China ‡ Institute of Plant Protection, Gansu Academy of Agricultural Sciences, No. 1 Nongkeyuan New Village, An’ning District, Lanzhou 730070, P. R. China S Supporting Information *

ABSTRACT: Because of its relatively high water solubility and mobility, 2,4-dichlorophenoxy acetic acid (2,4-D) has a high leaching potential threatening the surface water and groundwater. Controlled release formulations of 2,4-D could alleviate the adverse effects on the environment. In the present study, positive-charge functionalized mesoporous silica nanoparticles (MSNs) were facilely synthesized by incorporating trimethylammonium (TA) groups onto MSNs via a postgrafting method. 2,4-D sodium salt, the anionic form of 2,4-D, was effectively loaded into these positively charged MSN-TA nanoparticles. The loading content can be greatly improved to 21.7% compared to using bare MSNs as a single encapsulant (1.5%). Pesticide loading and release patterns were pH, ionic strength and temperature responsive, which were mainly dominated by the electrostatic interactions. Soil column experiments clearly demonstrated that MSN−TA can decrease the soil leaching of 2, 4-D sodium salt. Moreover, this novel nanoformulation showed good bioactivity on target plant without adverse effects on the growth of nontarget plant. This strategy based on electrostatic interactions could be widely applied to charge carrying agrochemicals using carriers bearing opposite charges to alleviate the potential adverse effects on the environment. KEYWORDS: mesoporous silica nanoparticles, 2,4-D sodium salt, electrostatic interaction, controlled release, soil leaching, bioactivity

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bentonites,7,8 and organo-palygorskite9 are the most extensively investigated materials. Adsorption and controlled release of 2,4D from granule matrix formulations based on lignins,10 activated carbon,11 and biochars12 have also been studied. Ethylcellulose as a microsphere matrix and gelatin−arabic gum complex as an envelope for CRFs of 2,4-D was elaborated by double encapsulation using a solvent evaporation technique followed by the complex coacervation method.13 In addition to physical encapsulation or absorption in polymeric matrix, chemical combination of 2,4-D with polymers through covalent bonding provides another useful strategy for controlled release of active ingredient. The 2,4-D was chemically caged by coupling with photoremovable protecting groups of coumarin or perylene-3-ylmethanol derivatives, and controlled release of 2,4-D was achieved by irradiating the caged compounds using UV−vis light.14−16 Since Mobil’s discovery of MCM-41,17 research on mesoporous silica nanoparticles (MSNs) has gained worldwide interest due to MSNs’ remarkable properties, such as low cost,

INTRODUCTION The extensive use of pesticides in agriculture has contributed significantly to farmers’ income and food productivity. However, depending on the mode of application and environmental conditions, more than 90% of the applied pesticides are either lost in the environment or unable to reach the target area required for pest control, which not only increases the cost but also brings about adverse impacts on the environment.1 The 2,4-dichlorophenoxy acetic acid (2,4-D) is one of the most commonly used herbicides worldwide for postemergence control of broad-leaf weeds due to its low cost and good selectivity.2 Because of its relatively high water solubility, 2,4-D exists predominantly in anionic form and is weakly retained by soil particles.3 Therefore, it has a high leaching potential threatening surface water and groundwater particularly if heavy rains occur shortly after herbicide application.4 To overcome this concern, the development of controlled release formulations (CRFs) of 2,4-D, especially with nanomaterials as carriers, could be advantageous because CRFs allow the use of minimal amounts of herbicide for the same period of activity, which will reduce the leaching potential and environmental pollution.5 The performance of CRFs on controlling the release of herbicide is closely related to the carrier materials. Many natural inorganic and organic polymers have been used to prepare 2,4D CRFs. Clay minerals including layered double hydroxides,3 cationic surfactant-modified Arizona montmorillonites6 and © 2017 American Chemical Society

Special Issue: Nanotechnology Applications and Implications of Agrochemicals toward Sustainable Agriculture and Food Systems Received: Revised: Accepted: Published: 6594

April 27, 2017 June 14, 2017 June 22, 2017 June 22, 2017 DOI: 10.1021/acs.jafc.7b01957 J. Agric. Food Chem. 2018, 66, 6594−6603

Article

Journal of Agricultural and Food Chemistry

purchased from J&K Scientific Ltd., Beijing, China. Tetraethyl orthosilicate (TEOS, 99%) was purchased from Fluorochem Ltd., Hadfield, U.K. All other chemicals and reagents were commercially available and used without further processing. Deionized water was obtained from a Milli-Q water system (Millipore Corporation, Bedford, MA) and was utilized for all reactions and treatment processes. Synthesis of Positive-Charge Functionalized MSN (MSN-TA). The MSN-TA sample with a hexagonal well-ordered pore structure was synthesized from pristine MSN (P-MSN) by postgrafting during a two-step preparation. The first step of P-MSN synthesis used a sol−gel method reported by Radu with minor modifications.34 Briefly, 3.0 g of CTAB was dissolved in 2000 mL of water, and then 10.5 mL of 2.0 M sodium hydroxide was slowly introduced into the CTAB solution at room temperature under constant stirring with the stirring rate of 800 r/min. The mixture was heated to 80 °C in an oil bath, and then 15.0 mL of TEOS was added dropwise. The solution was stirred vigorously for 6 h at 80 °C. The white solid that formed during the process was collected, washed several times with ethanol and water, and dried at 80 °C overnight in an oven. To remove the surfactant, the as-synthesized white powder was calcined at 550 °C for 5 h. Positive-charge functionalization used a postgrafting synthesis according to amino-functionalized mesoporous silica with a little modification.35 Specifically, 0.5 g of P-MSN was resuspended in 20 mL of anhydrous toluene solution. After vigorous stirring for 30 min, the 2.0 mL of N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride solution (50% in methanol) was added. The resulting mixture was refluxed for 4 h under vigorous stirring. Samples were collected by centrifuging at 10 000 rpm for 10 min, washed, and redispersed with deionized water and ethanol several times. The nanoparticles were dried at 80 °C overnight in an oven. Characterization. Fourier transform infrared (FTIR) spectroscopy was conducted on a spectrometer (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA) with a potassium bromide pellet and recorded over the spectral region of 400 to 4000 cm−1 at a spectral resolution of 4 cm−1. Thermogravimetric analysis (TGA) was performed with a PerkinElmer Pyris Diamond (Woodland, CA) from 30 to 550 °C at 20 °C/min under a N2 atmosphere. The specific surface area and pore characteristics of the samples were studied by determining the nitrogen adsorption using a specific surface area and pore size analyzer (TriStarII 3020, Micromeritics Instruments Corp, Norcross, GA) at −196 °C. The sample was outgassed at 10−3 Torr and 120 °C for about 6 h prior to the adsorption experiment. From the adsorption data, the Brunauer− Emmett−Teller (BET) equation36 was used to calculate the specific surface area at a relative pressure (P/P0) of 0.06−0.22; the Barrett− Joyner−Halenda (BJH) model37 was used to estimate the pore size distribution from the desorption branches of the isotherms. The morphology and particle size of the prepared samples were characterized using scanning electron microscopy (SEM, SU8000, Hitachi Ltd., Tokyo, Japan, operated at 10 kV) and transmission electron microscopy (TEM, Tecnai G2, F20 S-TWIN, FEI, Oregon, with an accelerating voltage of 200 kV). For SEM observations, the samples were gold-plated and dried under vacuum prior to imaging. The average particle size was determined by statistical analysis of the SEM images of more than 200 nanoparticles. For TEM analysis, specimens were prepared by dispersing the as-obtained powder in water and then placing a drop of the suspension onto carbon-coated copper grids with air drying. The zeta potential measurements were performed using distilled water as a solvent on a ZetaSizer Nano ZS Analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, U.K.). The samples were prepared at 1 mg mL−1 to make intensities in the range suitable for scattering. Different pH values were adjusted by the addition of 0.01 M HCl or NaOH. Before measurement, each sample was ultrasonicated for 5 min to prevent any aggregation. X-ray photoelectron spectroscopy (XPS) was conducted on a photoelectron spectrometer (ESCALab 250Xi, Thermo Fisher Scientific) using 150 W monochromatic Al Kα radiation (1486.6 eV, 500 μm spot size) as the excitation source; all binding energies were

facile preparation, biocompatibility, large specific surface area, tunable pore size for high loading capacity, and ability for targeted and controlled release of cargo molecules with surface functionalization and polymer coatings.18−20 Taking advantage of these unique properties, MSNs have attracted widespread interest and are an ideal scaffolding for delivery systems. A slow release formula of biological pesticide pyoluteorin was prepared using mesoporous silica as carriers, giving an example of putting an unstable compound inside the pores to avoid its fast degradation.21 The insecticide imidacloprid was effectively loaded into unmodified MSNs for termite control, and the effect of pore size, specific surface area, and mesoporous structure on uptake and release of biocide was systematically studied.22 Prado reported nanosized silica modified with carboxylic acid as a support for the controlled release of the herbicides 2,4-D and picloram.23 For controlled release in response to external stimuli, a novel redox-responsive decanethiol gatekeeper was grafted onto MSNs to mediate the delivery of salicylic acid with glutathione reducing agent.24 Other pesticides, such as metalaxyl,25 tebuconazole,26 vancomycin,27 and essential oil components28 were physically or chemically combined with MSNs for controlled release. Recently, we have developed quaternized chitosan-capped mesoporous silica nanoparticles as nanocarriers for controlled pyraclostrobin release.29 Noncovalent (e.g., hydrophobic, hydrogen bonding, and ionic) interaction between cargo molecules and carrier material mainly affect the loading capacity and release profile.30,31 Although research on the controlled release of pesticides using MSNs as carriers has had some progress, the driving forces for pesticide loading and release in most studies are hydrogen bonding and hydrophobic interactions. Ionic interactions are the long-range interactions that involve the electrostatic attraction and repulsion between oppositely charged ions. These are studied less and therefore are poorly exploited as a tool for achieving satisfactory loading and controlled release of pesticides. Taking full advantage of ionic interactions, positive charge functionalized MSNs have been used for drug and gene delivery.32,33 For charge carrying pesticide molecules, the loading content should be expected to increase by strengthening the electrostatic attraction through a modification of the surface of MSNs to bear more opposite charges. During the pursuit of ideal carrier for controllable loading and release of 2,4-D to alleviate the adverse effects on the environment, positive charge functionalized MSNs have to be ideal carriers for 2,4-D sodium salt, the anionic form of 2,4-D. In the present study, we report the synthesis of positively charged MSN (MSN-TA) by incorporating trimethylammonium (TA) functional groups onto the pristine MSN (P-MSN) and the adsorption of 2,4-D sodium salt into MSN-TA samples. The parameters that impact loading content such as the ratio of carrier to pesticide, temperature, pH value, various anions, as well as the release profiles were studied. Furthermore, the ability to decrease soil leaching was studied in comparison to the free pesticide 2,4-D sodium salt. Finally, we investigated the bioactivity of this nanosized CRFs of 2,4-D sodium salt against one dicot target plant cucumber (Cucumis sativus L.) and one monocot nontarget plant wheat (Triticum aestivum L.).

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MATERIALS AND METHODS

Materials. N-Trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (50% in methanol), cetyltrimethylammonium bromide (CTAB, 99%), and 2,4-D-sodium salt monohydrate (98%) were 6595

DOI: 10.1021/acs.jafc.7b01957 J. Agric. Food Chem. 2018, 66, 6594−6603

Article

Journal of Agricultural and Food Chemistry calibrated by the C 1s peak of the surface adventitious carbon at 284.8 eV. Loading of 2,4-D Sodium Salt into MSN-TA Samples. A typical procedure for loading 2,4-D sodium salt into MSN-TA samples followed the procedure reported by Lee et al. with minor modification.32 Specifically, about 30 mg of MSN-TA were dispersed in aqueous solution of 2,4-D sodium salt with different concentrations (1.0 mL) followed by another 4.0 mL of water. The suspension was stirred at room temperature for 4 h and centrifuged at 10 000 rpm for 10 min. The nanoparticles were collected and washed one time with 1 mL of water and were freeze-dried with a vacuum freeze-dryer under −40 °C. The 2,4-D sodium salt-loaded MSN-TA sample was denoted as 2,4-D sodium salt@MSN-TA. The amount of unloaded 2,4-D sodium salt in the supernatant and washes were determined using high-performance liquid chromatography (HPLC, 1200-DAD (diode array detector), Agilent, Santa Clara, CA). The loading content (LC) of 2,4-D sodium salt was determined by an indirect method. The difference between the amount of 2,4-D sodium salt initially employed and its content in the combined supernatant and washes was defined as the amount of pesticide entrapped. The LC (%) of 2,4-D sodium salt was calculated as follows: LC (%) = (weight of pesticide entrapped in nanoparticles/weight of nanoparticles) × 100%. The encapsulation efficiency (EE) of 2,4-D sodium salt was calculated as follows: EE (%) = (weight of pesticide entrapped in nanoparticles/initial weight of pesticide) × 100%. The HPLC operating parameters were as follows: Eclipse Plus C18 reversed-phase column (5 μm × 4.6 mm × 150 mm); column temperature, 30 °C; mobile phase, (acetonitrile/0.2% formic acid aqueous solution (V/V) = 75:25); flow rate, 1.0 mL/min; and DAD signals, 284 nm. In Vitro Release of 2,4-D Sodium Salt. About 20 mg of 2,4-D sodium salt@MSN-TA nanoparticles were dispersed in 2.0 mL of release medium in dialysis bags (MWCO 2000 g/mol). The dialysis bag was placed into 200 mL of release medium in a D-800LS dissolution tester (Tianjin University, Tianjin, China) at a stirring speed of 100 rpm. The release medium was water at different pH values via diluted HCl and NaOH. To study the effects of ionic strength on the release profiles, a NaCl aqueous solution (0.1 M with pH of 6.8) was used as a release medium. To study the temperature effect, pure water at different temperatures (20, 30, and 40 °C) was adopted as the release medium. The accumulative release profile of 2,4-D sodium salt was calculated by measuring the concentrations of 2,4-D sodium salt dissolved in the release medium at different times. To measure the concentration, 1.0 mL of release medium was withdrawn at a given time intervals for HPLC analysis followed by supplying the same volume of fresh release medium to ensure the same total solution volume. The accumulative 2,4-D sodium salt released was calculated according to the following equation:

contamination of leachates with soil particles. Before pesticide application, columns were conditioned by passing them with water using a flow rate of 1 mL min−1 controlled by a peristaltic pump. This was followed by pesticide soil leaching experiments. The 2,4-D sodium salt@MSN-TA or 2,4-D sodium salt were evenly dispersed in 500 mg of silica gel (corresponding to ∼20 mg active ingredient) and then were placed on the top of the columns. Another 1 cm of quartz sand was uniformly placed on top of the pesticide. Every 10 mL of column leachate in each experiment was collected, and 2,4-D sodium salt was determined using HPLC. The accumulative 2,4-D sodium salt leached was calculated according to the following equation: n

El =

Ve ∑i = 0 Ci + V0Cn mpesticide

mpesticide

× 100%

where El is the accumulative 2,4-D sodium salt leached (%) in regard to pesticide applied; Ve is the leached volume at every sample time (Ve = 10 mL); Cn (mg/mL) is the 2,4-D sodium salt concentration in leachate at sample time n. The mpesticide (mg) is the total amount of pesticide applied. The measurements were performed in triplicate. Bioactivity of 2,4-D Sodium Salt@MSN-TA Nanoparticles. The bioactivity of 2,4-D sodium salt@MSN-TA nanoparticles were assessed by means of a laboratory bioassay with one target dicot plant cucumber (Cucumis sativus L.) and one monocot nontarget plant wheat (Triticum aestivum L.). For cucumber bioassay experiments, 9 cm Petri dishes with a filter paper were used, according to the laboratory bioassay of fluorescent 2,4-D derivatives against Vigna radiate reported by Atta et al.14 In total, 15 similarly sized germinated seeds were placed in each Petri dish moistened with 10 mL of 2,4-D sodium salt@MSN-TA samples at a concentration equal to the field application rate (0.6 kg/ha). The control was similarly performed with the same amount of 2,4-D sodium salt free technical and pure distilled water. Each treatment was performed in triplicate. The Petri dishes were incubated in a light growth chamber with a 12 h photoperiod with a light intensity of 80 μmol photon m−2s−1 provided by fluorescent lamps. Day and night temperatures were set at 26 and 15 °C, respectively, and the humidity was kept at 80%. Each Petri dish was moistened with an equal volume of distilled water for daily watering. After 10 days of incubation, the root length and fresh weight were recorded to evaluate the bioactivity on the target plant. For the nontarget plant wheat, the bioactivity was tested on pots (10 cm high with diameter of 9.0 cm) filled with 240 g of soil, according to the bioassay of 2,4-D nanoformulations against Zea mays reported by Abigail et al.39 Six seeds of wheat were sown in each pot and grown in the greenhouse. The pots were watered daily with 10 mL of distilled water. One week after germination, the solution of 2,4-D sodium salt@MSN-TA samples was applied postemergence at an application rate of 2.5 kg/ha corresponding to the maximum application dose recommended for field application of 2,4-D.6 The same amount of free 2,4-D sodium salt technical and treatment without herbicide were used as controls. Each treatment was replicated three times. One week after herbicide application, the plant height and fresh weight of the aerial part of the wheat were determined to monitor nontarget plant responses to the nanoformulation. Statistical Analysis. Statistical analysis of the values was conducted using SPSS 10.0 (SPSS Inc., Chicago, IL) software. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The values are mean ± SD for three determinations in each group. P values ⩽0.05 were considered as significant.

n−1

Er =

Ve ∑i = 1 Ci

× 100%

where Er is the accumulative 2,4-D sodium salt released (%) in regard to loaded pesticide; Ve is the sampled volume taken at a predetermined time interval (Ve = 1.0 mL); Cn (mg/mL) is the 2,4-D sodium salt concentration in release medium at time n; V0 is the volume of release solution (200 mL). The mpesticide (mg) is the total amount of pesticide entrapped in the nanoparticles. The measurements were performed in triplicate. Soli Leaching Experiment. The performance of nanocarrier of MSN-TA in reducing the leaching of 2,4-D sodium salt was evaluated with leaching experiments through a soil column. The soil was collected locally from a depth of 0−15 cm, dried in the shade, ground to pass through a 2 mm sieve, and stored in polythene bags at room temperature. It has a pH of 7.2 and organic matter content of 4.0%. The composition was sand (85.3%), clay (1.7%), and silt (13%). Glass columns (20 cm length, 2.5 cm diameter) were packaged uniformly with air-dried soil, which occupied about 12 cm of the column.38 The top 2 cm were filled with quartz sand, and the bottom 2 cm with other quartz sand plus glass wool to minimize losses of soil and

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RESULTS AND DISCUSSION Preparation and Characterization of MSN-TA Nanoparticles. P-MSN was synthesized via a liquid crystal templating mechanism using CTAB as the structure-directing agent and TEOS as the silica source under basic conditions. Positive-charge functionalized MSNs (MSN-TA) were fabricated through a postgrafting strategy by incorporating the TA group onto P-MSN. Figure 1 shows the schematic illustration 6596


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Figure 1. Schematic illustration of the synthesis of MSN-TA nanoparticles.

Figure 2. SEM (A, B) and TEM (C, D, E) images of the TA groups functionalized MSN (MSN-TA). TEM images D and E were viewed along and perpendicular to the channel direction. Scale bars: (A) 5.0 μm, (B) 1.0 μm, (C) 0.5 μm, and (D, E) 50 nm.

of the synthesis of MSN-TA nanoparticles. The morphology of MSN-TA nanoparticles was observed using SEM and TEM (Figure 2). The SEM and TEM micrographs show that the assynthesized MSN-TA nanoparticles exhibited spherical morphology with a relatively smooth surface and an average particle size of about 423 nm. Particle diameters were estimated by statistical analysis of the SEM images of randomly selected 300 nanoparticles. The histograms of particle size distributions of MSN-TA are shown in the Supporting Information (Figure S1). Well ordered mesoporous structures with hexagonal arrays (Figure 2D) and straight lattice fringes (Figure 2E) can be seen when the electron beam is parallel and perpendicular to the pore axis, which is the characteristic of MCM-41-type MSN.40 Good monodispersity was achieved due to the electrostatic repulsion between positively charged particles from the introduction of quaternary ammonium groups. Incorporation of TA functional groups onto the P-MSN samples did not obviously affect the P-MSN’s morphology and size (data not shown). The FTIR spectra of P-MSN and MSN-TA are shown in Figure 3. The 1087 cm−1 broad absorption band found in PMSN and MSN-TA was attributed to characteristic Si−O−Si (siloxane) stretching vibrations. The absorption band at 1478 cm−1 in MSN-TA could be assigned to the C−H bending vibration of −N(CH3)3+, confirming the conjugation of TA groups on P-MSN. TGA is frequently used to study the thermal stability and decomposition pattern of chemicals and materials. Figure 4

Figure 3. FTIR spectra of P-MSN, MSN-TA, 2,4-D sodium salt, and 2,4-D sodium salt@MSN-TA.

displays TG analysis of P-MSN and MSN-TA. Calcined P-MSN is thermo-stable and maintains a constant weight in the temperature ranges studied here. About 15% weight loss is clearly seen in the TGA curve of MSN-TA, and this is mainly due to the decomposition of organic TA groups, which is further evidence that P-MSN was successfully grafted with positively charged TA groups. 6597


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Journal of Agricultural and Food Chemistry Table 1. Mesoporous Structure Characterization of Nanoparticlesa sample

SBET (m2/g)

Vt (cm3/g)

DBJH (nm)

P-MSN MSN-TA 2,4-D sodium salt@MSN-TA

1356.0 956.4 454.8

1.65 0.59 0.03

3.75 2.31

a

SBET, BET specific surface area; Vt, total pore volume; DBJH, BJH pore diameter.

blocked with the TA groups. However, the MSN-TA nanoparticles also have a narrow pore-size distribution with an average pore diameter of 2.54 nm according to Figure 6B. The adsorption of 2,4-D sodium salt into MSN-TA samples was related to extreme decreases in pore volume (0.59−0.03 cm3/g). This decrease occurred because the nanochannels were almost fully occupied by the pesticide molecules, which left little space for nitrogen adsorption. The zeta potential measures residual charges on the surface of nanoparticles. It is an indicator for the quaternary ammonium groups on the surface of MSN. The magnitude of the zeta potential is very important in determining the stability of nanoparticle systems. Generally, nanoparticles having the zeta potential values higher than +30 mV or lower than −30 mV are considered as stable systems.41,42 The presence of quaternary ammonium may produce higher zeta potential values. The zeta potential of P-MSN and MSN-TA samples in solutions at pH 3, 7, and 9 was measured. At pH 3.0, both the P-MSN and MSN-TA showed the positive zeta potential values of +7.2 and +67.8 mV, respectively. At pH 7.0, the zeta potential values for P-MSN and MSN-TA were −22.9 and +19.6 mV, respectively. At this neutral solution conditions, the silanol groups (Si−OH) on the surface of MSN became deprotonated, and thus the P-MSN exhibited negative zeta potential. The MSN-TA retained their positive zeta potential due to the high density of positively charged TA groups. At pH 9.0, all the samples showed negative zeta potential: −42.4 mV for P-MSN and −23.1 mV for MSN-TA. This pH-dependent zeta potential could explain the pH-responsive controlled release. Loading of 2,4-D Sodium Salt into MSN-TA Nanoparticles. With this MSN-TA carrier in hand, the LC and EE of 2,4-D sodium salt were next optimized including the solvent and ratio of carrier-to-pesticide. The LC and EE results of 2,4-D sodium salt under various conditions are presented in Table 2. The LC increased with increasing 2,4-D sodium salt. This is possibly due to the higher concentrations of 2,4-D sodium salt, which generated a strong gradient to facilitate the diffusion of cargo molecules into the MSN pores. The amount of 2,4-D sodium salt adsorbed onto the MSN-TA nanoparticles plateaued (up to 21.7%) when the mass ratio of pesticide to carrier reached 1:0, which implied a saturation absorption of 2,4-D sodium salt (entry 4, Table 2). The EE decreased with the increase of the mass ratio of cargo to carrier. When methanol was used as the solvent, the LC decreased from 21.7% to 14.5% (entry 6, Table 2) due to the higher solubility of 2,4-D sodium salt in water. It is very interesting that when PMSN instead of MSN-TA was used, the LC decreased sharply to 1.5% (entry 7, Table 2). At this neutral solution condition, the determined zeta potential value for P-MSN was −22.9 mV. As a consequence, the electrostatic repulsions between the negative charges and 2,4-D sodium salt lead to the prominent decrease of loading content. On the other hand, these results

Figure 4. TGA of P-MSN, MSN-TA, 2,4-D sodium salt, and 2,4-D sodium salt@MSN-TA.

XPS provides valuable information about the elements on the MSN surface. The XPS spectra given in Figure 5 display bands

Figure 5. XPS spectra of P-MSN (A) and MSN-TA (B).

assigned to elements in P-MSN and MSN-TA. In Figure 5A, binding energies of about 104.1 and 533.4 eV are assigned to the Si 2p and O 1s in P-MSN, respectively. The weak signal at 284.8 eV corresponds to the C 1s originated from the residual carbon after calcination for removing the template. Figure 5B shows the signals at 402.7, 286.2, and 197.6 eV corresponding to the N 1s, C 1s, and Cl 2p, respectively, which confirmed successful incorporation of TA functional groups onto P-MSN. The XPS results show that the atomic percentage of N is 3.27%. BET specific surface area and BJH pore size and volume analysis were used to explore the nanoparticles’ mesoporous structure. The values for the BET specific surface area (SBET), the total pore volume (Vt), and the BJH pore diameter (DBJH) are summarized in Table 1. Nitrogen adsorption−desorption isotherms and pore-size distribution of P-MSN and MSN-TA are shown in Figure 6. The P-MSN samples display a type IV isotherm curve with a sharp increase in volume adsorbed between 0.3 and 0.4 of P/P0 shown in Figure 6A, which is characteristic of a well-defined mesoporous structure. The BET specific surface area reduced from 1356.0 to 956.4 m2/g after the TA groups were grafted, while the pore volume decreased from 1.65 to 0.59 cm3/g suggesting that part of the pores were 6598


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Figure 6. Nitrogen adsorption−desorption isotherms of P-MSN, MSN-TA, and 2,4-D sodium salt@MSN-TA (A) and pore size distributions of PMSN and MSN-TA (B).

release, soil leaching, and bioactivity assay. The successful loading of 2,4-D sodium salt into MSN-TA was also confirmed by FTIR and TGA analysis (Figures 3 and 4). The effects of solution pH, ionic strength, and temperature on the LC were also studied. When inorganic salt NaCl or Na2SO4 was added to the solution, the LC of 2,4-D sodium salt decreased with increasing ionic strength, pH, and temperature (Figures S2−S4). The electrostatic interactions dominated the loading of 2,4-D sodium salt into MSN-TA nanoparticles under various conditions. Loading and release are opposite processes; higher LC means lower release. Thus, we next discussed the possible reasons underlying the stimuli-responsive loading and release patterns. Controlled Release of 2,4-D Sodium Salt. Pesticide carriers with controllable release in response to environmental stimuli are highly desirable for better efficacy and fewer side effects. The pesticide release profiles of the as-prepared positively charged MSNs were studied to reveal their potential in pesticide delivery system. The release profiles of 2,4-D sodium salt@MSN-TA samples under three different pH values of 3, 7, and 10 were investigated. Figure 7A shows that the release behaviors of 2,4-D sodium salt were obviously pHsensitive. The samples showed the lowest initial release in the

Table 2. Loading Content (LC) and Encapsulation Efficiency (EE) of 2,4-D Sodium Salt into MSN-TA Nanoparticlesa entry

solvent

mass ratiob

1 2 3 4 5 6 7c

H2O H2O H2O H2O H2O MeOH H2O

0.4 0.6 0.8 1.0 1.2 1.0 1.0

LC (%) 15.0 ± 0.3 17.5 ± 0.3 19.2 ± 0.2 21.7 ± 0.3 22.0 ± 0.4 14.5 ± 0.3 1.5 ± 0.1

EE (%) d c b a a

44.0 ± 1.2 35.3 ± 0.3 29.7 ± 0.4 27.7 ± 0.4 23.5 ± 0.6 17.0 ± 0.4 1.6 ± 0.1

a b c d e

a

MSN-TA (30 mg), H2O (5.0 mL), room temperature. Values are mean ± SD of three replicates. Values in each column followed by different letters are statistically different at P ⩽0.05 as determined by Duncan’s multiple range test. bMass ratio of 2,4-D sodium salt to MSN-TA. cP-MSN instead of MSN-TA was used.

clearly demonstrated the vital importance of electrostatic attractions for enhanced loading content. Considering the LC and EE together, the conditions of the cargo/carrier ratio of 1:1, pure water as solvent and room temperature (entry 4, Table 2) were adopted for scale preparation of 2,4-D sodium salt-loaded MSN-TA samples, which were used for the in vitro

Figure 7. 2,4-D sodium salt released at different pH values and ionic strengths (A) and temperatures (B). Error bars correspond to standard errors of triplicate measurements. 6599


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Figure 8. Schematic illustration of the controlled-release mechanism of an anionic pesticide 2,4-D sodium salt loaded in positive-charge functionalized MSNs. Mechanism A: pesticide released by electrostatic repulsion under neutral or basic solution. Mechanism B: Pesticide released through ion-exchange by increasing the ionic strength.

Figure 9. Breakthrough curves (A) and accumulative leaching profiles (B) for 2,4-D sodium salt applied for soil columns as MSN-TA formulations and free technical. Error bars correspond to standard errors of triplicate measurements.

first 2 h (16%) under pH 3.0, while the corresponding release rate was 43% under pH 10.0. The electrostatic interactions dominated the release profiles when the pH of the environment changed. The schematic illustration of the controlled-release mechanism of an anionic pesticide 2,4-D sodium salt is shown in Figure 8. In a weak acid solution, the MSN-TA nanoparticles carry more positive charge (zeta potential, +67.8 mV at pH 3.0), and a strong electrostatic attraction impeded the release of negatively charged 2,4-D sodium salt. At higher pH values, the silanol groups (Si−OH) in the MSN-TA nanoparticles are deprotonated, and a strong electrostatic repulsion between the negative charges of SiO− groups and negative charges of 2,4-D sodium salt would increase the release rate (mechanism A, Figure 8). The effect of ionic strength on the release profiles was also studied. Figure 7A shows that when a NaCl solution (0.1 M)

was used as a release medium, the release rate was faster than in pure water (red line in Figure 7A). The release profile has an obvious burst release. About 90% of the 2,4-D sodium salt was released after only 5 h; the release was only 40% in pure water at the same time interval. This ionic strength-triggering release is mainly due to the ion-exchange mechanism. The competitive electrostatic attraction between negatively charged chloride ions and positively charged TA groups would compel 2,4-D sodium salt to be far away from the TA group centers (mechanism B, Figure 8). This definitely facilitated payload release. We also studied thermoresponsive cargo molecule delivery because it is one of the most common stimuli-responsive strategies. Figure 7B shows that the release of 2,4-D sodium salt was temperature-dependent. More 2,4-D sodium salt was released at higher temperatures. At 40, 30, and 20 °C, the 6600


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Figure 10. Bioactivity for target plant cucumber (Cucumis sativus L.) determined in terms of root length and fresh weight: (A) control without treatment, (B) free 2,4-D sodium salt technical, and (C) 2,4-D sodium salt@MSN-TA samples. Bars marked with different letters are statistically different at P ⩽0.05 as determined by Duncan’s multiple range test.

Figure 11. Bioactivity for nontarget plant wheat (Triticum aestivum L.) determined in terms of plant height and fresh weight: (A) control without treatment, (B) free 2,4-D sodium salt technical, and (C) 2,4-D sodium salt@MSN-TA samples. Bars marked with different letters are statistically different at P ⩽0.05 as determined by Duncan’s multiple range test.

eluent for 2,4-D CRFs and free technical, the maximum leaching amount was greatly reduced from 3.7 to 1.7 mg. When the cumulative volume of 340 mL eluent was applied, the total amount of 2,4-D sodium salt leached was clearly lower in the CRFs (48.4%) than in the free system (97.3%) (Figure 9B). The soil column leaching test confirmed controlled release of 2,4-D sodium salt from formulations based on MSN-TA nanoparticles. This retarded the vertical movement of the herbicide through soil and reduced the leaching potential. Bioactivity of 2,4-D Sodium Salt@MSN-TA Samples. The bioactivity of the nanoformulations was compared to free herbicide and pure water using one dicot target plant cucumber (Cucumis sativus L.) and one monocot nontarget plant wheat (Triticum aestivum L.). The 2,4-D sodium salt nanoformulations were statistically as effective as the free herbicide in controlling

accumulative releases of 2,4-D sodium salt were 96, 75, and 52%, respectively, after 900 min. These temperature-controllable release patterns could possibly occur via the well-known temperature-dependent, diffusion-controlled process. High temperature may facilitate diffusion of payloads from the pores of MSN to the release medium. Retarded 2,4-D Sodium Salt Leaching in Soil. The results of the soil column experiments are seen in breakthrough curves (BTCs) in which the amount of 2,4-D sodium salt leached (milligram) in each collected fraction is shown as the ordinate in relation to the cumulative volume of eluent applied presented as the abscissa. The BTCs of the 2,4-D sodium salt@ MSN-TA samples and free 2,4-D sodium salt technical as control are shown in Figure 9A. Although the breakthrough of 2,4-D sodium salt occurs under the same cumulative volume of 6601



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of the test plant cucumber (Figure 10), when 2,4-D sodium salt at a concentration equal to the field application rate (0.6 kg/ha) was applied. There was obvious root length inhibition. Ten days after application, the fresh weight was reduced to about 50% compared to treatment without herbicide, demonstrating good herbicidal bioactivity. Slight lower inhibition effect of 2,4D sodium salt nanoformulation than free herbicide indicated the controlled release at the first stage. 2,4-D is a selective, systemic herbicide used for control of broad-leaved weeds. It is safe for monocot plant under recommended dosage. In the present study, wheat was selected as a model plant to evaluate the safety of 2,4-D sodium salt@ MSN-TA samples toward the monocot plant. At an application rate of 2.5 kg/ha corresponding to the maximum application dose recommended for field application of 2,4-D, both the nanoformulation and free 2,4-D sodium salt applied postemergency did not affect the plant height and free weight (Figure 11). Abigail et al. also reported that the nanoformulation of 2,4-D based on rice husk nanosorbents as carriers does not affect the development of nontarget plant (Zea mays).39 Therefore, the prepared CRFs of 2,4-D sodium salt can be satisfactorily used to control dicot plant without injuring the monocot plant. In summary, a stimuli-responsive controllable anionic pesticide release system has been designed by incorporating positive charges on the surface of MSN. Electrostatic interactions are the driving forces that facilitate pesticide loading. This regulates release and decreases soil leaching potential. Good bioactivity was seen on the target plant with no impact on the nontarget plant. Hence, positively charged MSNs used as nanocarriers for 2,4-D sodium salt could reduce environmental pollution without affecting bioactivity. The strategy based on electrostatic interactions could be widely applied to charge-carrying agrochemicals using carriers bearing opposite charges to alleviate the potential adverse effects on the environment.

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ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01957. Distribution of particle size of MSN-TA nanoparticles and effects of solution pH, ionic strength, and temperature on the loading content of 2,4-D sodium salt into MSN-TA nanoparticles (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/fax: +86-10-6281-6909. ORCID

Lidong Cao: 0000-0001-7217-7102 Qiliang Huang: 0000-0001-9820-7218 Author Contributions ∥

REFERENCES

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S Supporting Information *

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Article

L.C. and Z.Z. contributed equally to this work.

Funding

This work was supported by the State Key Development Program for Basic Research of China (Grant No. 2014CB932204) and the National Natural Science Foundation of China (NSFC) (Grant No. 31471805). Notes

The authors declare no competing financial interest. 6602


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Positive-Charge Functionalized Mesoporous Silica Nanoparticles as

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