*Article* β**-Cyclodextrin-Modified Mesoporous Silica Nanoparticles with Photo-Responsive Gatekeepers for Controlled Release of Hexaconazole**

**Hua Pan † , Wenjing Li † , Litao Wu, Weilan Huang and Fang Zhang \***

> Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; panh@emails.bjut.edu.cn (H.P.); liwenjing9511@126.com (W.L.); wltuiao@163.com (L.W.); huangweilan1226@163.com (W.H.)

**\*** Correspondence: zhangfang2000@bjut.edu.cn

† These authors contributed equally to this work.

**Abstract:** In the present research, photo-responsive controlled-release hexaconazole (Hex) nanoparticles (Nps) were successfully prepared with azobenzene (Azo)-modified bimodal mesoporous silica (BMMs), in which β-cyclodextrin (β-CD) was capped onto the BMMs-Azo surface via host– guest interactions (Hex@BMMs/Azo/β-CD). Scanning electron microscopy (SEM) confirmed that the nanoparticles had a spherical structure, and their average diameter determined by dynamic light scattering (DLS) was found to be 387.2 ± 3.8 nm. X-ray powder-diffraction analysis and N<sup>2</sup> -adsorption measurements indicated that Hex was loaded into the pores of the mesoporous structure, but the structure of the mesoporous composite was not destroyed. The loading capacity of Hex@BMMs/Azo/β-CD nanoparticles for Hex was approximately 27.3%. Elemental components of the nanoparticles were characterized by X-ray photoelectron spectroscopy (XPS) and electron dispersive spectroscopy (EDS). Ultraviolet–visible-light (UV–Vis) absorption spectroscopy tests showed that the azophenyl group in BMMs-Azo undergoes effective and reversible *cis-trans* isomerization under UV–Vis irradiation. Hex@BMMs/Azo/β-CD Nps exhibited excellent light-sensitive controlledrelease performance. The release of Hex was much higher under UV irradiation than that in the dark, which could be demonstrated by the bioactivity test. The nanoparticles also displayed excellent pH-responsive properties, and the sustained-release curves were described by the Ritger–Peppas release kinetic model. BMMs nanocarriers had good biological safety and provided a basis for the development of sustainable agriculture in the future.

**Keywords:** nanoparticle; hexaconazole; photo-responsibility; mesoporous silicas; controlled-release

#### **1. Introduction**

Pesticides are considered the most effective way to control pests and diseases in farmland, and improve agricultural productivity [1]. Currently, traditionally formulated pesticides are dominant in China. However, more than 90% of the pesticides cannot accurately act on specific targets, and their effective utilization rate is low due to poor water dispersibility, dust drift, volatilization, evaporation, and degradation, which has led to serious environmental pollution [2–4]. With the rise of nanotechnology in the agricultural field, slow- and controlled-release pesticides have been rapidly developed [5–7]. Environmental stimulus-responsive systems, such as pH- [8–10], redox- [11,12], temperature- [13,14], enzyme- [15,16], and ultraviolet (UV)-light-responsive [17,18] materials have been developed for triggered pesticide release, which can improve the utilization rate of pesticides and reduce environmental pollution.

Among the various stimulative-responsive systems, light-responsive materials are particularly attractive and more achievable in practical applications [19,20]. UV light, as an external stimulative factor, is widely used in intelligent nano slow/controlled-release

**Citation:** Pan, H.; Li, W.; Wu, L.; Huang, W.; Zhang, F. β-Cyclodextrin-Modified Mesoporous Silica Nanoparticles with Photo-Responsive Gatekeepers for Controlled Release of Hexaconazole. *Coatings* **2021**, *11*, 1489. https://doi.org/10.3390/ coatings11121489

Academic Editor: Yu Shen

Received: 14 October 2021 Accepted: 22 November 2021 Published: 2 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tion.

[12,24].

systems, due to its good controllability, remote control, and clean energy. Unlike the traditional "gatekeeper" system, a light-response system is reversible [21]. Pesticides can be accurately released under the illumination of a specific light source at the specified location. accurately released under the illumination of a specific light source at the specified loca-Azobenzene (Azo) is a novel UV-light-responsive molecule, and shows *trans/cis-*iso-

Among the various stimulative-responsive systems, light-responsive materials are particularly attractive and more achievable in practical applications [19,20]. UV light, as

ditional "gatekeeper" system, a light-response system is reversible [21]. Pesticides can be

*Coatings* **2021**, *11*, x 2 of 15

Azobenzene (Azo) is a novel UV-light-responsive molecule, and shows *trans/cis*isomers. It has been reported that *trans-* and *cis*-isomers of azobenzene (Azo) can be reversibly converted between each other. *Trans-to-cis* isomerization can occur under UVlight (300–400 nm) irradiation, while *cis-to-trans* isomerization can be induced by visible light (435 nm) or heating [22,23]. The host–guest interaction of *trans-*Azo and β-cyclodextrin (β-CD) was well recognized by hydrophobic and van der Waals forces, while *cis*-Azo cannot be, which is fully reversible. No side reaction or decomposition after long-term irradiation occurs, which is one of the ideal photochemical reactions in the materials application field [12,24]. mers. It has been reported that *trans-* and *cis-*isomers of azobenzene (Azo) can be reversibly converted between each other. *Trans-to-cis* isomerization can occur under UV-light (300–400 nm) irradiation, while *cis-to-trans* isomerization can be induced by visible light (435 nm) or heating [22,23]. The host–guest interaction of *trans-*Azo and β-cyclodextrin (β-CD) was well recognized by hydrophobic and van der Waals forces, while *cis-*Azo cannot be, which is fully reversible. No side reaction or decomposition after long-term irradiation occurs, which is one of the ideal photochemical reactions in the materials application field Bimodal mesoporous silica (BMMs) with a double-channel structure was synthesized

Bimodal mesoporous silica (BMMs) with a double-channel structure was synthesized by the sol–gel method [25,26]. Compared with traditional silicon mesoporous materials, the large pore volume of BMMs can significantly improve the loading capacity of pesticides. BMMs are widely used as drug carriers due to their adjustable structure, good stability, excellent biocompatibility, and low toxicity [27,28]. Hexaconazole (Hex) is a triazole fungicide with broad-spectrum protection and is used as a treatment for plant diseases caused by fungi [29,30]. However, the usage of Hex is limited due to its poor water solubility and low utilization efficiency. In addition, Hex has a long residual life in the environment and is very difficult to degrade in the agricultural soil and water environment. The residual Hex reduces soil and water quality, and causes irreversible harm and toxicity to soil microorganisms and animals and plants in water [31,32]. Therefore, it is desirable to design and prepare an intelligent, controlled-release system to improve the utilization rate and biological safety of Hex. by the sol–gel method [25,26]. Compared with traditional silicon mesoporous materials, the large pore volume of BMMs can significantly improve the loading capacity of pesticides. BMMs are widely used as drug carriers due to their adjustable structure, good stability, excellent biocompatibility, and low toxicity [27,28]. Hexaconazole (Hex) is a triazole fungicide with broad-spectrum protection and is used as a treatment for plant diseases caused by fungi [29,30]. However, the usage of Hex is limited due to its poor water solubility and low utilization efficiency. In addition, Hex has a long residual life in the environment and is very difficult to degrade in the agricultural soil and water environment. The residual Hex reduces soil and water quality, and causes irreversible harm and toxicity to soil microorganisms and animals and plants in water [31,32]. Therefore, it is desirable to design and prepare an intelligent, controlled-release system to improve the utilization rate and biological safety of Hex. Herein, aminoazobenzene modified with a silane coupling agent was grafted onto

Herein, aminoazobenzene modified with a silane coupling agent was grafted onto the surface of BMMs, and Hex was encapsulated in the mesopore surface through physical adsorption. A host–guest supramolecular valve was formed after *trans*-Azo was recognized by β-cyclodextrin (β-CD), which was further wrapped on a BMMs carrier to prepare the light-responsive pesticide-release system (Hex@BMMs/Azo/β-CD). The procedure is shown in Figure 1. The physico-chemical characteristics, release behavior, and biological activity of nanoparticles (Nps) were investigated. This study provides a new method of improving the utilization efficiency of pesticides and reducing environmental pollution. the surface of BMMs, and Hex was encapsulated in the mesopore surface through physical adsorption. A host–guest supramolecular valve was formed after *trans-*Azo was recognized by β-cyclodextrin (β-CD), which was further wrapped on a BMMs carrier to prepare the light-responsive pesticide-release system (Hex@BMMs/Azo/β-CD). The procedure is shown in Figure 1. The physico-chemical characteristics, release behavior, and biological activity of nanoparticles (Nps) were investigated. This study provides a new method of improving the utilization efficiency of pesticides and reducing environmental pollution.

**Figure 1.** Schematic diagram for the preparation of Hex@BMMs/Azo/β-CD and its light-responsive **Figure 1.** Schematic diagram for the preparation of Hex@BMMs/Azo/β-CD and its light-responsive release mechanism.

#### **2. Materials and Methods**

#### *2.1. Materials*

release mechanism.

Hexaconazole (95%) was purchased from Beijing Jinyue Biotechnology Co., Ltd. (Beijing, China). P-aminoazobenzene, 3-isocyanatopropyltriethoxysilane, and β-cyclodextrin (β-CD) were supplied by Sigma Aldrich (Saint Louis, MO, USA). Cetyltrimethy-lammonium

bromide (CTAB), tetraethyl orthosilicate (TEOS), and ammonia (25%, analytical reagent grade) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Anhydrous tetrahydrofuran (THF) was purchased from Tianjin Fuchen Chemical Reagent Factory (Tianjin, China). N-hexane was purchased from the Beijing Chemical Plant (Beijing, China). Deionized water was obtained with a Milli-Q water purification system (Millipore Co., Burlington, MA, USA) and used for all reactions and treatment processes in the laboratory.

#### *2.2. Preparation of BMMs*

CTAB (1.0448 g) was dissolved in 41.6 mL of distilled water in a flask and TEOS (3.2 mL) was slowly added dropwise to the solution. Subsequently, 0.96 mL of ammonia was quickly added into the flask with continued stirring until the solution turned into a white gel. The white gel was then suction-filtered, washed with distilled water, and dried at 100 ◦C for 8 h. The obtained product was heated to 550 ◦C at 5 ◦C/min for 5 h to remove CTAB to attain BMMs.

#### *2.3. Modification of Azo-Si*

Isocyanate propyltriethoxysilane (1.0255 g), p-aminoazobenzene (0.794 g), and anhydrous tetrahydrofuran (THF, 7.5 mL) were added to a three-necked flask. The reaction mixture was stirred at 70 ◦C for 10 h under a nitrogen atmosphere. To obtain Azo-Si, 40 mL of nhexane was used as a precipitator. After THF was removed by rotary evaporation, the solid precipitate was frozen overnight at −20 ◦C and collected by vacuum filtration. The product was washed with n-hexane and dried at 35 ◦C for 8 h to yield orange needle-like crystals.

### *2.4. Preparation of BMMs/Azo Nps*

After drying at 110 ◦C for 2 h, 300 mg of BMMs were dispersed in 30 mL of anhydrous methanol and sonicated for 10 min. Next, 30 mg of Azo was added into the BMMs solution with nitrogen protection. The final reaction mixture was stirred at 65 ◦C for 12 h, and then filtered. The resulting yellow solid precipitate was washed with THF and methanol, and dried in a vacuum at 35 ◦C for 10 h to provide the product (BMMs/Azo).

#### *2.5. Preparation of Hex@BMMs/Azo Nps*

BMMs/Azo (100 mg) and hexaconazole (20 mg/mL) were added to a flask, and the resulting solution was stirred at 35 ◦C for 24 h, centrifuged at a speed of 5000 rpm, washed with methanol three times, and dried at 35 ◦C for 8 h. The obtained products were denoted Hex@BMMs/Azo Nps.

### *2.6. Preparation of Hex@BMMs/Azo/β-CD Nps*

Hex@BMMs/Azo (100 mg) was immersed in phosphate buffer solution (PBS, pH = 7.4), and then 50 mg of β-CD was added. The mixture was placed in the magnetic stirrer for 24 h. Next, the solution was centrifuged for 8 min at 5000 rpm, rinsed with PBS three times, and dried at 35 ◦C for 8 h to obtain H@BMMs/Azo/β-CD Nps.

#### *2.7. Azo Loading Capacity*

Nps were collected by centrifugation, and the concentrations of Azo were detected by high-performance liquid chromatography (HPLC, 1200 Series, Agilent Corp., Wilmington, DE, USA) with a C18 bonded reverse-phase column. The mobile phase of the methanol– water mix (70:30, *v/v*) was programmed with a flow rate of 1.0 mL/min at 210 nm with a UV detector. The injection volume was 10 µL and the retention time was 12 min at 30 ◦C. The loading capacities (LC) were calculated as follows:

$$L\mathcal{C}(\%) = \frac{\mathbb{C}\_{loading} \times V\_{loading} - \mathbb{C}\_{residual} V\_{residual} - \mathbb{C}\_1 V\_1 - \mathbb{C}\_2 V\_2 - \mathbb{C}\_3 V\_3}{m}$$

where *LC* is the Hex loading capacity (%), *m* is the total amount of Nps (mg), *Cloading*, *Cresidual*, and *C*1, *C*2, *C*<sup>3</sup> are the Hex concentrations of the original loading, residual, and eluted solutions for the first, second, and third time respectively (mg/mL) and *Vloading*, *Vresidual*, *V*1, *V*2, and *V*<sup>3</sup> are the volumes of the original loading, residual, and eluted solutions for the first, second, and third time, respectively (mL).

#### *2.8. Characterization of Hex@BMMs/Azo/β-CD Nps*

The morphologies and the electron dispersive spectroscopy (EDS) maps of nanoparticles were observed by scanning electron microscopy (SEM) (Zeiss, Oberkochen, Germany). Particle size, polydispersity index (PDI), and zeta potential were analyzed by a dynamic light scattering (DLS) detector (Malvern Instruments Ltd., Malvern, UK). The obtained nanoparticles were suspended in deionized water at pH 7.0 ± 0.1.

The structure and interaction analyses of samples were conducted using X-ray powder diffraction (XRPD, D8 ADVANCE X, Bruker/AXS, Inc., Karlsruhe, Germany) and Fourier-transform infrared spectroscopy (FTIR, Nicolet Nexus 470, Nicolet Instrument Corp., Concord, CA, USA). The nitrogen adsorption–desorption isotherms were determined using an Autosorb-iQ pore analyzer (Quantachrome, Boynton Beach, FL, USA). The surface areas, pore-size distributions, and pore volumes were calculated using the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda methods. Thermogravimetric analyses (TGA) were carried out by a thermal analyzer (PerkinElmer, Waltham, MA, USA). Elements were analyzed by using X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Fisher Scientific, Waltham, MA, USA). Photoisomerization of azophenyl groups was measured using a UV–Vis spectrophotometer (UV-2600 Shimadzu Co., Ltd., Tokyo, Japan) at wavelength of 250–600 nm.

#### *2.9. Adhesion Test*

The contact angles of Hex@BMMs/Azo/β-CD Nps on leaves were measured using a contact-angle apparatus (JC2000D2M, Powereach Ltd., Shanghai, China). Technical Hex, Hex@BMMs/Azo, and deionized water were used as controls. Two-week-old tomato leaves were selected as model plants, and carefully washed with deionized water. After the leaves were air-dried, Hex@BMMs/Azo/β-CD dispersion, Hex@BMMs/Azo dispersion, technical Hex solution, and water were dropped onto the leaves on a glass slide, respectively. Images of the droplet on the leaf surface were taken, and the corresponding contact angles were detected using a contact-angle meter (Dataphysics-TP50, DataPhysics Instruments, Filderstadt, Germany).

#### *2.10. Release Behavior of Hex@BMMs/Azo/β-CD Nps*

Hex@BMMs/Azo/β-CD Nps (15 mg) were suspended in 3 mL of methanol–water solvents (50:50, *v/v*). The suspension was transferred to a dialysis bag (molecular cutoff capacity 3500 kDa) and then put into flasks with 60 mL of methanol/H2O (1:1, *v/v*) solution. The release medium was then shaken at a speed of 100 rpm at 37 ◦C and irradiated continuously with 150, 300, and 500 W UV lamps (365 nm). Five milliliters of the sample were collected at fixed time intervals, and the same amount of fresh medium was replenished in the system. Hex contents were measured by HPLC, and the release ratio of Hex from the nano-delivery sample was calculated.

To investigate the pH sensitivity of Hex, the release of Hex from Nps at different pH levels (4.0, 7.0, and 9.0) under 300 W, 365 nm UV light was measured using the same method as described above. The cumulative release rate of Hex in Nps was then calculated using the following equation:

$$Q(\%) = \frac{V\_0 \times \mathcal{C}\_T + V \times \sum\_{N=1}^{T-1} \mathcal{C}}{W} \times 100\%$$

where *W* is the total mass concentration of Hex in the Pickering emulsion, *V*<sup>0</sup> is the volume of the sustained-release solution, and *V* is the volume of the slow-release solution taken at

a specific interval time. *C<sup>T</sup>* and *C* are the concentrations of *T* and *N* samples, respectively. The release kinetics of Hex from Hex@BMMs/Azo/β-CD Nps was separately investigated using four mathematical models, namely the pseudo-zero-order equation, pseudo-firstorder equation, Higuchi model, and Ritger–Peppas model.

#### *2.11. Bioactivity Test*

Hex@BMMs/Azo/β-CD Nps (40 mg) were dispersed in 8 mL of sterile distilled water with 0.1% Tween 80, and then 4 mL of the aqueous suspension was exposed to UV light (365 nm) under stirring at 25 ◦C. Technical Hex solution, non-irradiated Nps, and distilled water were used as controls. *Rhizoctonia solani* cakes (5 mm in diameter) were placed into the center of potato dextrose agar (PDA) petri dishes with Nps (50, 100, and 200 mg/L). The dishes were incubated for 14 days at 28 ◦C and each colony diameter was measured every 24 h. The relative inhibition rates against the fungi were calculated according to the colony diameters.

#### *2.12. Biosafety Evaluation*

Human embryo skin fibroblast cells (CCC-ESF-1 cells) were seeded in 96-well plates in triplicate and cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦C for 24 h. Then, CCC-ESF-1 cells were treated with different concentrations (0, 31.25, 62.5, 125, 250, and 500 µg/mL) of BMMs/Azo/β-CD for 24 h, and the cell viability was evaluated using a cell counting kit (CCK8, Dojindo, Japan). *E. coli* was cultured in LB culture medium with different concentrations (0, 31.25, 62.5, 125, 250, and 500 µg/mL) of BMMs/Azo/β-CD Nps at 37 ◦C for 24 h, and the *E. coli* concentrations were determined by UV–Vis absorptiometry at 600 nm. All tests were carried out at least in triplicate.
