β-Glucan-Functionalized Mesoporous Silica Nanoparticles for Smart Control of Fungicide Release and Translocation in Plants.

In this work, an enzyme-responsive nanovehicle for improving captan (CAP) contact fungicide bioactivity and translocation in plant tissues was synthesized (CAP-MSNs-β-glucan) by attaching β-glucan to the outer surface of mesoporous silica nanoparticles. CAP-MSNs-β-glucan properties were tested by FTIR, ζ-potential, DLS, XRD, TGA, FE-SEM, and HR-TEM. Cargo protection ability of CAP-MSNs-β-glucan from photolysis and hydrolysis was examined in comparison to CAP commercial formulation (CAP-CF). CAP-MSNs-β-glucan distribution in plant tissues, bioactivity against Fusarium graminearum, and biotoxicity toward zebrafish (Danio rerio) were tested and compared with that of CAP-CF. CAP-MSNs-β-glucan results showed good loading efficacy reaching 18.39% and enzymatic-release dependency up to 83.8% of the total cargo after 20 days of β-glucan unsealing. CAP-MSNs-β-glucan showed significant release protection under pH changes. MSNs-β-glucan showed excellent CAP protection from UV. CAP-MSNs-β-glucan showed better distribution in corn tissues and 1.28 more inhibiting potency to Fusarium graminearum than CAP-CF. CAP-MSNs-β-glucan showed 1.88 times lower toxicity than CAP-CF to zebrafish after 96 h of treatment. We recommend using such formulations to overcome shortcomings of contact fungicides and achieve better and sustainable farming.

Introduction

Huge amounts of fungicides are applied globally to protect crops from fungal diseases. Traditional fungicide formulations are based on adhesives and adjuvants, which do not have selective or interactive release ability in the presence of the targeted pest.[1,2] Recently, the use of eco-friendly pesticides has become a global trend to maximize pesticide employment and minimize the potential risk to the environment and humans.[3−6] Many controlled release formulations (CRFs) have been reported previously. Although those formulations have excellent release behavior and high loading capacity, they do not offer control on where and when pesticide release will happen.[7−10] As a hopeful alternative, modified mesoporous silica nanoparticles (MSNs) offer incomparable characteristics as pesticide carriers, such as high stability, biocompatibility, simple fabrication, high loading efficiency, protection of nontargeted microorganisms from pesticide unintentional release, and its ability to connect triggering materials on their exteriors that operate as gatekeepers to control loaded pesticide release.[11−14] Furthermore, smaller MSNs can be delivered into vegetative vascular tissues, circumventing selective absorbability of roots.[15] Between all the innovative materials utilized in CRFs,[3,16,17] materials that have sensitivity to enzymes can be considered as the most suitable capping materials for allowing pesticides to be released from the nanoparticles only when specified pests are present.[6,12,18−20]

Plants have many defense mechanisms against pathogens, one of which is releasing polysaccharide hydrolyzing enzymes to destroy the invading pathogen cells.[21] β-1,3-Glucanase is considered one of these proteins—their production increases when plants are invaded. There are many published works supporting the fact that β-1,3-/1.4-/1,6-glucanases and chitinases play a defensive role by weakening microbial growth by hydrolyzing the chitin and glucan components in the pathogen cell walls.[22,23] The major cell-wall demolishing enzymes released by pathogens are arabinosidase; β-1,4-xylanase; cellulase; β-1,6-, β-1,4-, and β-1,3-glucanases; arabanase; and β-1,4-galactanase.[24] β-Glucan is a polysaccharide that exists in many organisms’ cell walls.[25]

The plant pathogen Fusarium graminearum is a critical pathogen for most grass species around the world, and it is responsible for stalk and ear rot of corn and head blight in wheat. Fusarium graminearum can cause different diseases in vegetative and generative organs of the plant, and it can infect corn plants in many stages of growth.[26,27]Fusarium graminearum produces mycotoxins that accumulate in affected plant tissues, and these mycotoxins represent a significant risk to human and animal health when they enter the food and feed chain.[28,29]

Captan is a broadly used phthalimide contact fungicide for the control of mildew, rots, blotches, and scabs on vegetables and fruits. Captan (CAP) has different impacts on fungal biochemistry that involve inhibition of DNA and enzyme synthesis based on electrophilic interactions with thiols in fungal enzymes and inhibition of respiration processes in numerous species of fungi and bacteria.[30,31] CAP has many disadvantages which need to be overridden such as lower translocation ability between plant tissues, high acute toxicity to nontargeted organisms, and fast hydrolysis and photolysis.[32] CAP showed many adverse effects on rainbow trout (Oncorhynchus mykiss). Fish tissues showed inflammation in the kidney trunk and necrosis in the liver. The highly impacted organs were the liver, the kidney trunk, and the gill.[33−35] According to the Federal Water Pollution Control Act, CAP has been labeled as a hazardous material under section 311(b)(2)(A) of (https://pubchem.ncbi.nlm.nih.gov/source/hsdb/951).

Our key strategy in addressing the weaknesses of CAP and controlling the filamentous fungus Fusarium graminearum is using the β-glucanase enzyme produced from the diseased plant as a cargo release trigger as well as decreasing CAP hazardous effects to the nontargeted organisms in the environment. Furthermore, using CAP in a smart release formulation would provide an effective defense to various plant tissues and achieve sustainable agriculture.

In this work, we synthesized β-glucanase stimuli-responsive nanocarriers by using MSNs as a vehicle and derivatized β-glucan as a release stopper. Our idea is to utilize the nonselective absorbability of 32 nm MSNs into the plant roots and the sensitivity of β-glucan to β-glucanase enzyme from the pathogenic fungi to control CAP release only when plants are invaded by pathogens besides improving the translocation ability of CAP in different plant tissues as well as decreasing CAP undesirable damage to the nontargeted organisms in the environment. β-Glucan before and after carboxymethylation has been characterized by XRD, FTIR, and TGA. CAP-loaded MSNs capped with β-glucan (CAP-MSNs-β-glucan) nanoparticle properties have been identified by FE-SEM, HR-TEM, FT-IR, ζ-potential, and dynamic light scattering (DLS). CAP-MSNs-β-glucan loading capacity has been tested by thermogravimetric analysis (TGA). CAP release behaviors from CAP-MSNs-β-glucan under different pH levels have been investigated. Also, the release behavior with the β-glucanase enzyme in the medium has been examined. The protection ability of MSNs-β-glucan for the trapped CAP from UV photolysis was examined in comparison to the CAP commercial formulation WP 50% (CAP-CF). The bioactivity of CAP-MSNs-β-glucan against stalk and ear rot (Fusarium graminearum) relative to that of CAP-CF has been evaluated in this work. CAP-MSNs-β-glucan distribution in corn plant tissues under field conditions relative to that of CAP-CF at different time periods has been assessed. Additionally, CAP-MSNs-β-glucan biotoxicity to zebrafish (Danio rerio) compared to CAP-CF has been tested.

Materials and Methods

Materials

Tianjin Sinos Biochemical Technology Co., Ltd. supplied captan (97%). Hebei Guanlong Agrochemical Co., Ltd. supplied captan wettable powder (50%). Shanghai Macklin Biochemical Co., Ltd. provided (3-aminopropyl)triethoxysilane (APTES) (98%) and tetraethyl orthosilicate (TEOS) (98%). Sigma-Aldrich Co. LLC provided 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) (98%), N,N-dimethylformamide (99.5%), β-glucanase enzyme (β-glucanase activity of 1 unit may liberate 1.0 M glucose in 1.0 h), and 2000 MWCO (D2272) dialysis tubing. β-Glucan (70%) (molecular weight 2.4 × 106 Da), N-hydroxysuccinimide (NHS) (98%), sodium chloroacetate (98%), isopropyl alcohol (99.5%), and hexadecyltrimethylammonium bromide (CTAB) (99%) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Tianjin Fuyu Fine Chemical Co., Ltd. provided ethanol (99.7%) and acetone (99.5%). Guangzhou Chemical Reagent Factory provided the toluene (99.5%). To create filtered water, a purification system (Likang Biomedical Technology Holdings Co., Ltd., Taiwan, China) was utilized.

Characterization

The chemically active groups were recognized by FT-IR spectrometer Vertex 70 (BRUKER, Germany). A Zetasizer Nano ZSE particle sizer (Malvern, UK) was used to analyze the ζ-potential and particle size. X-ray diffraction patterns (XRD) of β-glucan and carboxymethylated β-glucan were obtained using a XRD Rigaku diffractometer (Ultima IV model) (Rigaku,Tokyo, Japan) with a copper lamp radiation source (Cu Kα = 1.5406 Å), 20 mA current, 40 kV tension, and 8°/min speed. The morphology and structure of MSNs and CAP-MSNs-β-glucan were seen and characterized by a field emission scanning electron microscope Verios 460 (SEM, Thermo Fisher, USA) and a transmission electron microscope Talos F200S (TEM, Thermo Fisher, USA). Gold dust was applied to all SEM samples. A thermogravimetric analyzer TG209F1 LibraTM (Netzsch Instrument Manufacturing Co., Ltd., Germany) was used to determine the loading efficiency of CAP into the MSNs and analyze the thermogravimetric (TG) and the derived thermogravimetric (DTG) of β-glucan and carboxymethylated β-glucan. CAP distribution in plant tissues and in vitro release behavior were investigated by employing a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan) with a UV detector. Eclipse XDB-C18 column has been used. A volume of 10 mL was set as the injection volume for all standards and samples. At a flow rate of 1 mL min–1, the mobile phase was a mixture of two mobile phases A and B (85:15). The mobile phase (A) was methanol + 0.1% formic acid + 0.05% ammonia, and mobile phase (B) was water + 0.1% formic acid + 0.05% ammonia. The column temperature and the detection wavelength were 45 °C and 225 nm, respectively. All samples have been filtered with a 0.22 μm nylon syringe filter before being examined by HPLC.[36−38] The HPLC standard curve and typical chromatograms for CAP are presented in Figure S1 and S2 in the Supporting Information.

MSNs Synthesis and NH2 Surface Modification

MSNs were synthesized with an average size of approximately 32 nm according to the previous publications with our modifications.[15] Briefly, 10.2 g of KH2PO4 and 1.74 g of NaOH were dissolved in 1500 mL deionized water. The mixture was then given 55.65 g of CTAB. The obtained combination was agitated at 25 °C for 3 h to create a homogeneous CTAB solution. Following the complete dissolution of CTAB, 27.9 mL of TEOS was gently dripped into the mix, which then was agitated at 550 rpm and 25 °C for 8 h. Next, the obtained white residue was centrifuged and washed three times with an 80% ethanol solution. The obtained MSNs were dehydrated in a vacuum oven for 12 h. Then, the obtained MSNs were placed in a furnace at 600 °C for 5 h to burn out the template. Next, in 50 mL of toluene, a half-gram of the obtained template-free MSNs was suspended and kept under reflux at 110 °C with magnetic stirring. After 30 min, 1 mL of APTES was added. The same conditions were maintained for 24 h. The surface-modified MSNs-NH2 were centrifuged, washed with 80% EtOH aqueous solution, and dehydrated in the oven at 60 °C.

β-Glucan Derivatization

The β-glucan carboxymethylation process was performed according to the previous publications with our enhancements.[39] Ten grams of β-glucan was mixed with 120 mL of isopropyl alcohol and 12.6 mL of NaOH solution (7.5 mol L–1). At 10 °C, the mix was continuously stirred for 1 h; 3.95 g of sodium chloroacetate dispersed in 14 mL of deionized water was dropped into the mixture to generate sodium chloroacetate β-glucan. The temperature was raised to 70 °C and stirred for 2 h. Then, 6 mol L–1 hydrochloric acid solution was added to neutralize the excessive NaOH, and the resulting salts were eliminated by dialyzing the obtained residue for 72 h in deionized water.[40] The nondialyzed residual portion was lyophilized. Finally, in 15 mL acetone containing 5 g of dry sodium chloroacetate β-glucan, 30 mL of 6 mol L–1 HCl solution was added to convert sodium chloroacetate β-glucan to β-glucan-COOH. The mix was held with stirring for half an hour.[39] The resulting β-glucan-COOH was cleaned multiple times with a washing solution of methanol:water (4:1) and dried in a vacuum at 50 °C. FT-IR, TGA, and XRD analysis have all been used to verify the β-glucan carboxymethylation process.

CAP Loading and CAP-MSNs-β-glucan Preparation

Three-tenths of a gram of CAP was dissolved in 10 mL acetone, followed by adding 0.2 g of MSNs-NH2 in the mixture. For 24 h, the mixture was held with stirring followed by centrifugation to remove the acetone to obtain CAP-loaded MSNs (CAP-MSNs-NH2). The CAP-MSNs-NH2 were vacuum-dried at 50 °C for 6 h. Following that, 0.2 g of β-glucan-COOH and 0.3 g of EDC were mixed with 50 mL of pure water. The resulting mixture was stirred for 30 min at room temperature. Finally, 0.3 g NHS and 0.5 g CAP-MSNs-NH2 were mixed with 5 mL water and the obtained mix was poured into the reaction flask. The reaction was held with agitation for 24 h. The attained CAP-MSNs-β-glucan was cleaned to remove any extra interactants by washing with water and acetone three times, followed by vacuum drying for 24 h at 50 °C. To calculate the amount of CAP loaded into the MSNs-β-glucan, 5 mg of MSNs, MSNs-β-glucan, and CAP-MSNs-β-glucan were analyzed by TGA.

Determination of CAP Controlled Release Profile in Vitro

The release profiles of CAP at different pH levels or under stimuli-responsive release have been studied. For 20 days, in 500 mL of buffer solution, 0.3 g of CAP-MSNs-β-glucan was placed in a dialysis bag. The utilized buffers to evaluate CAP release were 0.1 mol L–1 KH2PO4 for pH 7, 0.1 mol L–1 acetate buffer for pH 4, and 0.2 mol L–1 Tris + 0.1 mol L–1 HCI for pH 9. To reach the sink condition and improve CAP dispersion, Tween 80 was added as 1% from the release medium. Acetone percentage was 10% of the release solution. The test was conducted in darkness, and the temperature was set at 25 °C. In the CAP release test in response to β-1,3-d-glucanase enzyme, β-1,3-d-glucanase (1 mg mL–1) was added at pH 7 at 3 days after starting the test. After different time intervals, 1 mL aliquots from the tested liquid were collected, filtered, and injected into the HPLC system to detect CAP levels.[37]

Protection from UV Photolysis

The cargo protective ability of MSNs-β-glucans from UV light was compared with that of CAP-CF. Two tenths of a gram of CAP-CF or the equivalent amount of CAP-MSNs-β-glucans and CAP-MSNs was combined with a 100 mL acetone–H2O mix (1:3, v:v) in a cylindrical reaction vessel (250 mL) supplied with a continuous stirring bar. A UV lamp (254 nm) was set 20 cm away from the experimental vessels. At different time intervals, 1 mL of the tested solution was collected, centrifuged, and injected into the HPLC system.

Bioactivity toward Fusarium graminearum

The bioactivity of CAP-MSNs-β-glucan, CAP-CF, CAP-MSNs, and technical CAP against Fusarium graminearum was tested. A series of concentrations increasing from 0.22 to 3.4 mg L–1 of CAP-MSNs-β-glucan, CAP-CF, or technical CAP were mixed with sterilized potato dextrose agar (PDA). Then, 5 mm mycelial plugs from the margins were cut from active Fusarium graminearum colonies and centered on the PDA media. The same doses of acetone solvent were inserted into PDA medium as a control. All PDA media were set at 25 °C in darkness. Finally, growth inhibition was calculated after the control diameter grew to 7 cm. Pathogenic inhibition was determined by the following equation:[41]EC50 values of CAP-MSNs-β-glucan, CAP-CF, and technical CAP against Fusarium graminearum were determined using GraphPad Prism 9.0 (San Diego, CA, USA).

CAP Delivery to Different Tissues in Corn Plant

CAP distribution behavior in corn plant tissues after seeds were treated with CAP-MSNs-β-glucan or CAP-CF was investigated under field conditions. The corn species Zea mays L. was utilized to perform the experiment in a free CAP trial zone. The trial zone was separated into nine sections, and each section was approximately 2 m2. Each trial was performed three times. The treated soils have been sterilized before starting the test. Technical CAP was prepared and applied to seeds based on previous publications,[42,43] with 0.62 g of technical CAP applied to 1 kg seeds. CAP-MSNs-β-glucan and CAP-CF concentrations were adjusted in corresponding amounts to the employed amount of technical CAP. The control trials were treated with water only. Samples were collected at different time periods from the roots, stalks, and leaves within 14 days after reaching the second leaf collar stage (V2). CAP was extracted from tissues by QuEChERS method and determined using HPLC.[44]

Acute Toxicity Test toward Danio rerio

In nonchlorinated water, the obtained zebrafish (3 ± 0.5 cm) have been placed. The conditions were set at 23 °C, pH 7.2, 90–100% dissolved oxygen, 12:12 h light:dark, and 120–150 mg L–1 CaCO3 as the total hardness. Commercial fish food was supplied to the fish. The 203 OECD Guidelines has been followed to perform the acute toxicity assay of CAP-MSNs-β-glucans or CAP-CF.[45] The tested concentrations of CAP-MSNs-β-glucans or CAP-CF in the fish media were 100, 200, 400, 600, 800, 1000, and 1200 μg L–1. Each testing solution consisted of 4 L, with 20 zebrafishes in each treatment. All the tests were done three times. The experiment lasted 96 h in static systems with no medium renovation. To improve the CAP-MSNs-β-glucan distribution, Tween 80 (1%) and sodium lignosulfonate (1%) were mixed to the release medium. Blank MSNs-β-glucans and blank water solutions were used as controls. The mortality rates were assessed after 24, 48, 72, and 96 h, and the LC50 was established.

Data Analysis

The data has been statistically analyzed by SPSS 26.0 software (SPSS, Chicago, IL, USA). EC50 values were estimated by GraphPad Prism 9.0 (San Diego, CA, USA). To statistically show all data, the mean ± standard deviation was utilized.

Results and Discussion

X-ray Diffractometry of β-Glucan

Figure A shows the diffractogram profiles of β-glucan and carboxymethylated β-glucan. The two diffractograms showed a significant difference. At the peak between 5° (2θ) and 15° (2θ), there was a minor shift in carboxymethylated β-glucan sample compared to β-glucan sample. After carboxymethylation, a new peak appeared in the area between 10° (2θ) and 17° (2θ), probably because of the carboxymethylated chemical bonds on the β-glucan polymer structure that could change β-glucan crystallinity. Additionally, the presence of peaks (27.44°, 31.78°, 45.44°, 56.54°, 66.44°, and 75.38°) indicates that there are changes in the β-glucan crystalline nature. According to the previous publication, the obtained diffractograms indicate that the β-glucan structure has been modified following carboxymethylation.[46]

Figure 1

X-ray diffraction (XRD) profiles of β-glucan and carboxymethylated β-glucan (A) FT-IR spectra of β-glucan (I) and carboxymethylated β-glucan (II) (B). TG and DTG curves for β-glucan (C) and carboxymethylated β-glucan (D).

FT-IR Analysis of β-Glucan and Carboxymethylated β-Glucan

Figure B shows the FT-IR spectra of carboxymethylated β-glucan and β-glucan. In both spectra, the bands at 3374 and 3380 cm–1 belong to the hydroxyl group (−OH) vibrations. Also, it can be noticed that the hydroxyl peak in the carboxymethylated β-glucan spectrum became wider due to the addition of (−OH) in the carboxymethylation step. The peak at 1653 cm–1 in the β-glucan spectrum belongs to the glucose ring.[47] The C–O–C vibrations (symmetric stretching) appeared at 1076 cm–1 in both spectra, while the asymmetric stretching vibrations appeared at 1251 and 1203 cm–1. In both spectra, the peak at 2924 cm–1 belongs to the C–H stretching in the glucose CH2 groups. At 1604 and 1421 cm–1 in the carboxymethylated β-glucan spectrum, the detected bands belong to asymmetric and symmetric vibrations of (COO−) and confirm the carboxymethylation step.

β-Glucan Thermal Characterization

Figure C,D show the TG and DTG curves of β-glucan and carboxymethylated β-glucan. TG data showed that β-glucan has lost weight through three phases. Water removal is responsible for the first stage of weight loss, which occurred up to 145 °C. Water loss is observed in the β-glucan DTG curve as an endothermic peak at 60.3 °C. The thermal breakdown of the molecule resulted in two weight loss points between 200 and 600 °C, with an exothermic peak detected in the DTG at 270.9 and 338.6 °C. The initial stage of carboxymethylated glucan weight loss occurred up to 145 °C and is ascribed to water removal with an endothermic peak at 61 °C in the DTG curve. Between 180 and 450 °C, the second stage of weight loss happened corresponding to the molecular breakdown which has been shown in the DTG curve with an exothermic peak at 266.2 °C. In both samples, the first weight loss is representing water loss, followed by the second stage of disintegration at 270 and 338.6 °C in β-glucan sample or at 266.2 °C in carboxymethylated β-glucan. Previous articles suggested that polysaccharide thermal stability could be reduced because of chemical groups addition.[48,49] The reduction of the thermal stability of the carboxymethylated-β-glucan confirms the carboxymethylation procedure.

Synthesis of CAP-MSNs-β-glucan

The formation steps of CAP-MSNs-β-glucan are shown in Scheme . Mesoporous silica nanoparticles (32 nm) were synthesized by condensing TEOS molecules on a CTAB template to generate a porous structure. The CTAB template was removed from the MSNs by burning at 600 °C. The surface hydroxyl groups (−OH) were modified to amino (−NH2) groups using APTES to obtain MSNs-NH2. Next, CAP was loaded into the MSNs-NH2 by immersing the MSNs-NH2 in CAP/acetone solution to obtain CAP-MSNs-NH2. On the other hand, β-glucan was reacted with sodium monochloroacetic acid to obtain a β-glucan derivative by replacing hydroxyl groups (−OH) in the glucose monomers with carboxyl groups (−COOH). Then, by interacting with hydrochloric acid, sodium carboxymethyl-glucan was transformed to carboxymethylated-glucan. Finally, in pure water with NHS and EDC, carboxymethylated glucan was used to cover CAP-MSNs-NH2 by establishing amide bonds (−CONH−) to obtain CAP-MSNs-β-glucan.

Scheme 1

Steps for Synthesizing CAP-MSNs-β-glucan

SEM and TEM Characterization

FE-SEM and HR-TEM were used to observe the structural properties of MSNs and CAP-MSNs-β-glucan. As presented in (Figure A), the examined MSNs in TEM images showed an identical structure, with an elliptical shape and an obvious mesoporous structure, as displayed in the SEM image in (Figure C). The obtained MSNs diameters ranged from 20.8 to 39 nm. (Figure B and D) shows CAP-MSNs-β-glucan under TEM and SEM, and there are differences observed in the outer morphology compared with MSNs. The outer structure of CAP-MSNs-β-glucan was modified and the mesoporous structure, as shown in (Figure A), was dimmed. These findings confirm that β-glucan are tightly attached to the MSNs exterior and have excellent dispersibility and a consistent shape.

Figure 2

TEM observations of MSNs (A) and CAP-MSNs-β-glucan (B). SEM observations of MSNs (C) and CAP-MSNs-β-glucan (D). (Gold dust was used to cover all SEM samples.)

Size Distributions and ζ-Potential

Figure A shows the blank MSNs and CAP-MSNs-β-glucan size distribution. The MSNs average size was around 32.7 nm, while the CAP-MSNs-β-glucan median size was approximately 78.8 nm. CAP-MSNs-β-glucan showed an increase in size because of the long length of the β-glucan chains.[50] It can be concluded that more than one MSN could be attached to a β-glucan chain, which led to an increase in the detected size. The ζ-potentials of MSNs-β-glucan, MSNs-NH2, and MSNs were measured. A negative charge was detected for the MSNs surface, and the ζ-potential was −7.6 mV. Following the surface modification with amino (−NH2) groups, the charge increased to +18.3 mV. Nevertheless, the MSNs-β-glucan ζ-potential has been reduced to −11.5 mV after conjugating β-glucan to MSNs exterior. The increase in the MSNs-NH2 surface charge could be attributed to the amino (−NH2) group effects, which eliminated the effect of the silanol (Si–O–H) group on the MSNs surface.[51] Furthermore, β-glucan hydroxyl (−OH) groups reduced the MSNs-β-glucan surface charge following β-glucan conjugation. The obtained results showed that the MSN surface has been modified and β-glucan was successfully attached.

Figure 3

MSNs and MSNs-β-glucans particle size distribution (A). N2 adsorption–desorption isotherms for MSNs and CAP-MSNs-β-glucans; insets show the pore size distribution of each MSN and CAP-MSNs-β-glucans (B). Fourier-transform infrared spectra of MSNs-β-glucan (I), MSNs-NH2 (II), and Blank MSNs (III) (C). Thermogravimetric curves for CAP-MSNs-β-glucan, MSNs-β-glucan, MSNs-NH2, and MSNs (D).

BET Analysis

Figure B displays the N2 adsorption–desorption isotherms for MSNs and CAP-MSNs-β-glucan. The obtained adsorption–desorption isotherm characteristics are presented in Table . The BET–BJH method was used to inspect the MSN mesoporous structure and pore size. All the isotherms revealed normal adsorption curves of type IV, which signifies a clear mesoporous structure.[52] Both N2 isotherms for MSNs and CAP-MSNs-β-glucan displayed hysteresis loops between relative pressures of 0.9 and 1. The BET surface areas of MSNs and CAP-MSNs-β-glucan were 925.1 m2 g–1 and 428.5 m2 g–1, respectively. Figure B inset shows the BJH pore size of blank MSNs and CAP-MSNs-β-glucan. The MSNs BJH pore size was 4.76 nm, which means that the material is mesoporous. However, the CAP-MSNs-β-glucan pore size was not detected. The BJH pore volume for MSNs and CAP-MSNs-β-glucan was 0.781 cm3 g–1 and 0.221 cm3 g–1, respectively. The obtained data confirm that CAP has been loaded into the pores of MSNs, and the modified β-glucans are strongly connected on the edges of the ordered mesopores leading to seal the MSNs pores.

Table 1

Blank MSNs and CAP-MSNs-β-glucan Characteristics Obtained from N2 Adsorption–Desorption Isotherms

Sample	SBET (m2·g-1)	BJH pore size (nm)	BJH Pore volume (cm3·g-1)
Blank MSNs	925.1	4.76	0.781
CAP-MSNs-β-glucan	428.5	-	0.221

FT-IR Spectroscopy

The FT-IR spectra of carboxymethylated-β-glucan, MSNs-β-glucan, MSNs-NH2, and pure MSNs are shown in Figure C. Normal mesoporous silica peaks were presented in the MSNs spectrum, for example, the stretching of the peaks of siloxane (Si–O–Si) at 811 and 1088 cm–1 and silanol at 3429 cm–1. After the amino groups were grafted onto the MSNs surface, the absorption peak of silanol group of MSNs nanoparticles was altered. In the MSNs-NH2 spectrum, the methylene (−CH2) group and the amino (−NH2) group from APTES appeared at 2957 and 1628 cm–1, which indicate MSNs surface grafting with (−NH2). After β-glucan conjugation, new peaks were observed at 1642 and 1560 cm–1, which belong to the amide bond (−CONH−). Moreover, a wider peak occurred at 3431 cm–1, and it is assigned to the hydroxyl groups (−OH) of β-glucan. Additionally, a stronger vibration of the methylene group (−CH2) peak occurred at 2935 cm–1, which is referring to the carboxymethylated β-glucan methylene group. All the obtained results confirm that the modified β-glucan was attached firmly to the MSNs surface.

CAP Loading Efficiency into the MSNs

TGA curves of CAP-MSNs-β-glucan, MSNs-β-glucan, MSNs-NH2, and MSNs are presented in Figure D. The weight loss was 4.62% for MSNs, 5.75% for MSNs-NH2, 17.83% for MSNs-β-glucan, and 36.22% for CAP-MSNs-β-glucan. The observed weight loss in the MSNs curve can be attributed to the ability of silica to absorb air humidity and release it at higher temperatures. However, the observed weight loss in the MSNs-β-glucan curve was due to the bound β-glucan on the MSNs outer shell. The weight loss pattern in MSNs-NH2 looks different from the MSNs weight loss, which could be attributed to surface modification steps that led to elimination of water residues from MSNs. It can be noticed that the weight loss in MSNs-NH2 starts at over 400 °C. The mass differences between MSNs-NH2 and MSNs can be attributed to the addition of APTES on the MSNs surface. Nonetheless, the mass differences between MSNs-β-glucan and CAP-MSNs-β-glucan were ascribed to the packed amount of CAP. The TGA data provide solid confirmation of the successful loading of CAP into MSNs-β-glucan. The precise CAP loading into the MSNs was 18.39% from the CAP-MSNs-β-glucan total weight. The results have been confirmed by HPLC, which showed that the loaded amount is 18.41% from CAP-MSNs-β-glucan total weight.

X-ray Diffractometry of MSNs

The crystalline structure of MSNs and MSNs-β-glucan has been tested by XRD. Figure A shows the diffractogram profiles of MSNs and MSNs-β-glucan. The MSNs sample showed a standard XRD pattern of mesoporous silica. The MSNs-β-glucan diffractogram showed a significant difference. At the peak between 15° (2θ) and 30° (2θ), there was a minor shift in the MSNs-β-glucan sample compared to the MSNs sample. After β-glucan conjugation to the MSNs surface, new peaks appeared. These peaks are probably caused by the addition of carboxymethyl β-glucan to the MSNs polymeric structure that could change the MSNs crystallinity. Additionally, the presence of peaks (45.24°, 56.26°, 66.02°, and 75.06°) that belong to carboxymethyl β-glucan as shown in Figure A, indicating that there are changes in MSNs crystalline nature and confirming the β-glucan conjugation.

Figure 4

X-ray diffraction (XRD) profiles of MSNs and MSNs-β-glucan (A). Influence of the presence or absence of β-glucanase on CAP release from CAP-MSNs-β-glucan starting from the 3rd day at 25 °C and pH 7 (mean  ±  SD, n = 3 for each sample) (B). Cumulative release profile of CAP-MSNs-β-glucan under different pH values (C).

CAP-MSNs-β-glucan Release Kinetics

CAP release behavior from CAP-MSNs-β-glucan in the presence of β-glucanase enzyme has been examined at pH 7 and 25 °C. Figure B displays the cumulative CAP release in the testing solution in the presence or absence of the β-glucanase enzyme. Until the third day and before adding the β-glucanase enzyme, 1 mg mL–1, into one of the dialysis bags in the dissolution tester, the CAP release was similar in both examination mediums. The release was changed from 3.94% to 7.87% from the 3rd day to the 20th day in the solution without enzyme. While in β-glucanase enzyme in the medium, the released CAP increased from 3.64% on the 3rd day to 83.87% on the 20th day. This result supported our hypothesis that CAP release occurred by selectively uncapping CAP-MSNs-β-glucan in the presence of β-glucanase enzymes by splitting 1,3-β-glycosidic bonds in β-glucan between β-d-glucose monomers. β-Glucanase enzymes can hydrolyze large β-linked polysaccharide glycosidic bonds, like β-glucan, generating glucose monomers. The discharge of CAP after pathogen invasion is the most vital stage, because Fusarium graminearum hyphae usually enter cereal floret tissues by direct penetration or by natural openings within 24 h of infection.[53] Enzymes that arestimulated upon the presence of fungal invasion, particularly β-glucanase enzymes, are the crucial key in liberating CAP from the CAP-MSNs-β-glucan.[54,55]

Figure C displays the CAP cumulative release in buffer solutions from CAP-MSNs-β-glucan at pH 5.0, 7.0, or 9.0. At pH 7.0, CAP cumulative release was the lowest, while at pH 5.0 and pH 9.0, the CAP release was higher. The released CAP from CAP-MSNs-β-glucan after 20 days reached 26.69% at pH 9.0, 7.18% at pH 7.0, and 16.42% at pH 5.0. An earlier work elucidated that β-glucan is tolerant to lower or higher pH.[56] However, the released CAP at pH 5.0 or pH 9.0 occurred for other circumstances. The release may have occurred at pH 5.0 due to amide (−CONH−) bond softness at lower pH, which led to the detachment of β-glucan from the MSNs surface and release of cargo.[12] On the other hand, the release at pH 9.0 occurred because of the instability of siloxane (Si–O–Si) bonding under high pH conditions, which led to disintegration of the MSNs structure.[57] Nevertheless, this work was consistent with other published works on other capping materials, and the β-glucan coating offered higher control over undesirable cargo release under pH changes.[58,59]

UV Photolysis

Table shows the UV radiation protective ability of CAP-CF, CAP-MSNs, and CAP-MSNs-β-glucans. CAP in CAP-CF was photolyzed faster than CAP in CAP-MSNs-β-glucans. After 24 h of UV radiation, CAP-CF showed 78.71% deterioration, while CAP from CAP-MSNs-β-glucans showed 16.93% degradation after the same elapsed period of radiation. CAP in CAP-MSNs showed 88.87% degradation after 80 h of exposure. These findings demonstrate that the prepared MSNs covered with β-glucans could provide excellent photolysis defense compared to CAP-CF.

Table 2

CAP Deterioration Ratio (%) by UV Radiation in CAP-CF, CAP-MSNs, and CAP-MSNs-β-glucansa

UV Irradiation	CAP-CF (%)	SD	CAP-MSNs (%)	SD	CAP-MSNs-β-glucans (%)	SD
0 h	0	0	0	0	0	0
3 h	30.02	3.59	23.51	2.22	7.09	4.52
6 h	52.73	5.91	37.64	5.97	10.68	4.24
12 h	63.94	5.88	43.91	4.77	12.45	2.44
24 h	78.71	2.43	59.23	2.56	16.93	6.28
36 h	84.68	6.10	63.51	5.56	21.30	5.62
40 h	89.55	5.08	67.16	4.71	34.51	3.45
60 h	95.32	4.48	81.49	4.89	36.07	5.83
80 h	100	0	88.87	3.04	48.01	3.83

Standard deviation (SD) (n = 3).

Bioactivity Test of CAP-MSNs-β-glucan

The EC50 values of technical CAP, CAP-CF, CAP-MSNs, and CAP-MSNs-β-glucan against Fusarium graminearum are shown in Table S1. The chosen concentrations stretched from 0.22 to 3.40 mg L–1. Fusarium graminearum inhibition was estimated when the growth of the control diameter reached 7 cm. The EC50 values for CAP-MSNs-β-glucan, CAP-CF, CAP-MSNs, and technical CAP were 0.445, 0.571, 0.652, and 0.666 mg L–1, respectively. The fungicidal activity of CAP-MSNs-β-glucan was found to be greater than that of CAP-CF, CAP-MSNs, and technical CAP. CAP-CF and technical CAP results could be ascribed to CAP’s great ability to hydrolyze to 4-cyclohexane, 1,2-dicarboximide, and carbon dioxide, which are not toxic to pathogens.[60] CAP-MSNs results could be ascribed to CAP release from the MSNs without any control leading to fast hydrolysis of the loaded CAP. Obviously, the shielding ability of MSNs-β-glucan, in contrast to CAP-CF, plays a role in protecting CAP from hydrolysis. MSNs-β-glucan may slow the degradation of CAP in the environment to nonbioactive byproducts and extend CAP efficiency without being released over a longer amount of time. Different growth inhibition of Fusarium graminearum induced by CAP-MSNs-β-glucan, CAP-CF, or technical CAP is presented in Figure .

Figure 5

Fungicidal activity of CAP-MSNs-β-glucans, CAP-CF, CAP-MSNs, and technical CAP against Fusarium graminearum.

Distribution of CAP in the Vascular System of Corn Plants

To investigate the plant tissue distribution of CAP-CF and CAP-MSNs-β-glucan in corn plants. Sample collection was started at the beginning of the V2-s leaf collar stage (7 days after planting). We focused on treating the tested seeds only with the formulations and avoided spraying the plant stalks or leaves. This technique can show the distribution behavior of CAP-MSNs-β-glucan through corn plant tissues, such as roots, stalks, and leaves. Figure depicts the distribution of CAP within 14 days following the V2-s leaf collar stage. The CAP concentration in CAP-MSNs-β-glucan-treated roots was greater after 14 days of treatment than in CAP-CF-treated roots. Moreover, CAP deterioration in leaves in the CAP-MSNs-β-glucan-treated group was slower than that of CAP-CF. Furthermore, CAP-CF decreased rapidly from day 1 to day 14. As displayed in Figure B and C, CAP-MSNs-β-glucan extended the presence of CAP in stalks and roots more than 14 days. These outcomes proved that CAP-MSNs-β-glucan could lead to significant concentrations of CAP being distributed into the different tissues for a longer time than with CAP-CF. In contrast, CAP-CF exhibited lower translocation into the different corn plant tissues. It was found that CAP concentrations in the tissues of CAP-CF-treated plants after 14 days from the V2 stage were 1.13 mg mL–1 in roots and 0.514 mg mL–1 in stalks. In leaves, CAP was undetectable. Nevertheless, CAP concentrations in the treated groups with CAP-MSNs-β-glucan were 20.62 mg mL–1 in roots, 11.99 mg mL–1 in stalks, and 5.45 mg mL–1 in leaves. These findings suggested that CAP-MSNs-β-glucan can keep and extend CAP bioactivity compared with the CAP-CF treatment. As a result, using CAP-MSNs-β-glucan might give significant protection to corn plants against a variety of pathogens in different tissues.

Figure 6

Distribution of CAP in different tissues of corn plants following treatment with CAP-MSNs-β-glucans vs CAP-CF. The tissue samples were taken at different times from roots (A), stalks (B), and leaves (C). Significant results were marked by (*) compared with CAP-CF.

CAP-MSNs-β-glucans Acute Toxicity to Danio rerio

CAP-MSNs-β-glucans biotoxicity to aquatic species was investigated and compared with CAP-CF, since CAP has been shown to have undesired side effects in fish.[61]Figure depicts the effects of CAP-MSNs-β-glucans and CAP-CF on the Danio rerio death rate at various concentrations and periods. The death rate increased steadily as concentration increased and time passed. After 96 h of CAP-CF exposure, most of the zebrafish perished in the upper concentration (1000 and 1200 μg L–1). The CAP-CF mortality rate at 1000 and 1200 μg L–1, after 24 h from the beginning of the test, reached 75% and 96.6%, respectively. Nevertheless, at the same concentrations, the death rate in CAP-MSNs-β-glucans was 55% and 90%. After 48 h, the death rate of zebrafish treated with CAP-CF at 800 μg L–1 was 53.3%, while at the same concentration, the zebrafish death rate in CAP-MSNs-β-glucans treatments was 33.3%. The death rate of zebrafish was higher after 72 h for CAP-CF treatment than CAP-MSNs-β-glucans treatment. In CAP-CF treatment, after 72 h the mortality rate rose very fast compared to the CAP-MSNs-β-glucans mortality rate. The LC50 for CAP-MSNs-β-glucans and CAP-CF treated zebrafish are shown in Table S2. CAP-MSNs-β-glucans and CAP-CF toxicity to zebrafish increased over time. After the first 24 h, CAP-MSNs-β-glucans LC50 was 1.97 times higher than CAP-CF. The LC50 of CAP-MSNs-β-glucans after 96 h of treatment was 1.88 times higher than CAP-CF. The achieved results signify that CAP-MSNs-β-glucans are safer than CAP-CF. The findings of this study shown that CAP-MSNs-β-glucans may substantially decrease CAP toxicity.

Figure 7

CAP-MSNs-β-glucans and CAP-CF dose-dependent effect on Danio rerio mortality.

Conclusion

In summary, by using 32 nm MSNs covered with β-glucan as a doorkeeper, we created β-glucanase enzyme-responsive controlled release formulation (CAP-MSNs-β-glucan). TEM and SEM results of CAP-MSNs-β-glucan revealed an excellent shape uniformity of the prepared MSNs. Also, XRD, FTIR, and TGA results confirmed that β-glucans have been carboxymethylated perfectly to work as a gatekeeper on the MSNs surface. The FTIR, TGA, and ζ-potential confirmed that β-glucan has conjugated on the MSNs surface. CAP-MSNs-β-glucan showed an outstanding loading efficacy reaching 18.39% and an enzymatic-release dependency up to 83.8% of the total CAP after 20 days of β-glucan unsealing. MSNs-β-glucans showed better UV protection to the trapped CAP than CAP-CF. CAP-MSNs-β-glucans showed significant release protection under pH changes. Also, the EC50 of CAP-CF and technical CAP against Fusarium graminearum was higher than that of CAP-MSNs-β-glucans. Moreover, under field conditions, CAP-MSNs-β-glucans presented better distribution in corn from roots to leaves than the CAP commercial product (CAP-CF). Moreover, CAP-MSNs-β-glucan showed 1.88 times lower toxicity than CAP-CF to zebrafish after 96 h of treatment. The obtained results confirm our hypothesis that MSNs-β-glucans can improve CAP activity and translocation inside the plant with lower environmental toxicity. We recommend using such smart release formulation to enhance fungicide bioactivity, decrease the environmental biotoxicity, and override the contact fungicide shortcomings.