Phosphorylated zein as biodegradable and aqueous nano-carriers for

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Agricultural and Environmental Chemistry

Phosphorylated zein as biodegradable and aqueous nano-carriers for pesticides with sustained-release and anti-UV properties Li Hao, Guanquan Lin, Chuangyu Chen, Hongjun Zhou, Huayao Chen, and Xinhua Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03060 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Journal of Agricultural and Food Chemistry

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Phosphorylated zein as biodegradable and aqueous nano-carriers for pesticides with sustained-release and anti-UV properties

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Li Hao a,b, Guanquan Lin a,b, Chuangyu Chen a, Hongjun Zhou a,b*, Huayao Chen a,b, Xinhua

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Zhou a,b*

1 2

6 7 8 9 10 11

aSchool

12 13 14 15 16

* Hongjun Zhou, Phone: +86-20-89003114, E-mail: [email protected] * Xinhua Zhou, Phone: +86-20-89003114, E-mail: [email protected]

of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, P. R. China; bKey Laboratory of Agricultural Green Fine Chemicals of Guangdong Higher Education Institution, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, P. R. China

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ACS Paragon Plus Environment


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ABSTRACT: Zein’s prevalent hydrophobic character is one of major challenges associated

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with ineffective utilization as aqueous nano-carrier for pesticides. Herein, we reported an

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effective approach to hydrophilic modification of zein by phosphorylation using nontoxic

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sodium tripolyphosphate (STP), thereby improving the water-solubility, foliage wettability

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and adhesion ability of zein as nano-carrier for sustained release of pesticides. The procedure

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relied on zein grafted with STP via N- and O- phosphate bonds and encapsulation of

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avermectin (AVM) as a hydrophobic model drug using phosphorylated zein (P-Zein), which

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achieved pH sensitivity to controlled release of AVM in various applicable environments.

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The chemical interaction between zein and STP was confirmed by Fourier transform infrared

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(FTIR), thermogravimetric analysis (TGA), and differential scanning calorimetric (DSC).

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Scanning electron microscope (SEM), dynamic light scattering (DLS), and zeta potential

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technique were applied to investigate their structural characteristics and stability, from which

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found that AVM encapsulated in P-Zein (AVM@P-Zein) formed uniform nanoparticles with

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average size in the range of 174-278 nm under different conditions, and had an excellent

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stability in aqueous solution. Besides, AVM@P-Zein facilitated the wettability on the foliage

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surface evidenced from contact angle values owe to the amphiphilic character after

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phosphorylation as well as enhanced the adhesion ability between liquid and leaf, restricting

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the pesticides run off. Ultraviolet-visible (UV-vis) spectroscopy was employed to explore the

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anti-UV property and encapsulation as well as release behavior, which revealed that the

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presence of P-Zein like a shell protect AVM from UV photolysis with encapsulation

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efficiency of approximately 81.52% and the release of AVM from P-Zein showed

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pH-responsive behavior ascribed to protonation and deprotonation of phosphate under 2


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various pH conditions fitting to Elovich kinetic model, achieving the relative more rapid

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release under acidic conditions. More importantly, AVM@P-Zein retained the toxicity for

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insecticidal effect.

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KEYWORDS: Phosphorylated zein; Nano-carrier; Foliage wettability; Sustained release; pH

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sensitivity

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INTRODUCTION

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As the excessive use of pesticides bring serious environmental pollution and human

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health problems, while causing the evolution of pest resistance, there are globally growing

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demands to develop approaches for improving the utilization efficiency of pesticides and

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avoiding foliar loss, oxidation, photolysis, etc., thus reducing the consumption and related

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side effects of pesticides.1 The demands are intensified in light of the fact that huge amounts

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of organic solvents are employed to disperse pesticides in applicable situation, hence waiting

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for more environmental-friendly, controlled-released, and aqueous-based means of

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pesticide-delivery system.2 The current efforts to controlled-deliver pesticides have faced

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several critical challenges associated with the incompatible carriers. For example, the initial

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burst release of pesticides usually lead to sharp decline below the effective level of active

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ingredients.3-4 The another concern is the residue of carrier materials due to nondegradable

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intrinsic character causing secondary pollution.

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Over the past few decades, nanotechnology based controlled release formulations have

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attracted much interests in the area of drug or pesticide delivery for improving utilization and

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preventing pollution by reducing leaching and volatilization.5-6 The main aim behind utilizing

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nano-carriers for active ingredient delivery is their size-specific unique properties with

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nano-size in the range of 10 nm to 200 nm and high surface area, which can contribute to

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deposit on plant leaves and reduce pesticide waste, thus enhance permeability and retention

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time, achieve better thermal stability, and also be especially benefit to improve water

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solubility.7-10 In light of such intriguing properties, researchers have investigated

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nano-carriers for drugs in medicine or pesticides in plant or crop field extensively across the

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globe recently, and developed several novel formulations, such as mesoporous silicon,

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polymeric nanocapsules, etc..11-13 Recently, IUPAC identified top ten emerging chemical

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innovations with sustainable potential around the globe, among which nanopesticides lies on

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top with the promising potential to change our world.14 The main challenges with

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nanomaterials are to address high drug loading, preventing premature release before reaching

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the desired site, as well as biodegradable due to the necessity of elimination of nanoparticle

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residues.15

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To this end, implementation of green nanotechnology and renewable materials as

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nano-carriers have recently received considerable attention. For instance, various natural

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source matrices, including protein, i.e. feather keratin, soy protein, biodegradable polymers,

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i.e. polylactic acid (PLA), polycaprolactone (PCL), and polysaccharides, i.e. chitosan,

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cellulose, alginate, were investigated and utilized intensively.16-22 Among this kind of

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substances, zein, as a major storage protein extracted from corn endosperm, possesses special

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tertiary structure with high hydrophobicity ascribed to a high proportion (＞50%) of nonpolar

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amino acids.23-25 Zein also exhibits the characteristics of good biodegradability,

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biocompatibility, high thermal resistance, and non-toxicity, and also have the ability to

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self-assemble into nanoparticles, which render them a potential biomaterial for the

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development of colloidal delivery system for various hydrophobic drugs or pesticides.26-27 By

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relying on these intriguing properties, existing literature offers some strategies for utilizing

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zein as vehicles in agriculture, food industry, and medicine field.28-29 Zein is derived from

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waste of corn starch using as carrier for pesticides or drugs to improve utilization. In contrast

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to nanocarriers using hydrophilic animal proteins, such as casein, silk protein, gelatin,

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collagen, etc., hydrophobic plant proteins such as zein have the capability of yielding

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sustained release. Also, zein with high hydrophobicity is cheaper than animal proteins and

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possesses functional groups which can be easily used, when modified with hydrophilic

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groups, it may form amphiphilic micelles to contain more hydrophobic pesticides or drugs.

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Oliveira et al.30 described zein nanoparticles using antisolvent precipitation method as

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eco-friendly carrier systems loaded with essential oil of citronella, which showed effective

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protection of the repellents against UV degradation. Scholten et al.31 prepared zein/tannic

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acid complex particles using anti-solvent precipitation technique, and reported the

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hydrophobicity of the particles controlled by the incorporation of various amounts of

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hydrophilic tannic acid. However, the main limitation and concern of zein applied in the field

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of pesticide is the need for the large amounts and cost of orgainic solvents, i.e. ethanol, owe

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to its water-insoluble intrinsic property.32 Thereby, it is imperative to develop novel

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approaches to address the poor water solubility of zein.

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Recently, phosphorylation has been widely used in the food industry in attempting to

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improve the functional properties of food protein, such as soybean, egg white, and milk

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protein based on thermal stability, gelation, water-holding capacity, and phosphate soluble

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ability, etc..33-35 For instance, Ma et al.36 fabricated ovalbumin emulsion modified by

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phosphorylation under dry-heating and demonstrated the introduction of phosphate groups

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enhanced the intermolecular interaction, made ovalbumin disperse into water better, and

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improved the stability of emulsion. Additionally, chemical modification is considered to be

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an efficient approach in changing the molecular structure of proteins and improving

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properties.37

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functional

Traditionally,

phosphorylation

with

hypertoxic

phosphorus

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oxychloride (POCl3) is a common method, but which existed the serious harm effect by

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producing hydrogen chloride while reacting in water.38 Sodium tripolyphosphate (STP)

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exhibits the advantages of safety, low cost, and simple operation as a kind of phosphorylation

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reagent, which is also proved by Food and Drug Administration (FDA) as a food additive.39-40

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Also, phosphates can provide nutritions for plants or crops.41 Such intriguing and promising

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properties render STP useful in improving water retention and intensifying the water-soluble

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ability of protein, further as nutrients agents to some extent. To date, there are rare literatures

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reported on zein modified with STP, and zein molecular structural transformation and

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interaction during phosphorylation modification have not been explored intuitively.

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Additionally, there are limited researches stated employing phosphorylated zein as

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nano-delivery system of AVM. Comprehensively, AVM encapsulated in phosphorylated zein

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can achieve synergistic effect as both insecticides and nutrients.

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In present work, we demonstrated zein can be hydrophilic modified by phosphorylation

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with STP, thereby improving the water-solubility, foliage wettability and water-holding

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capacity of zein as nano-carrier for sustained release of pesticides. AVM was selected as

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model pesticide due to its hydrophobicity and instability, such as easy to oxidation and

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photodegradation. Such AVM encapsulated in phosphorylate zein are specifically designed to

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release their payload response to pH ascribed to zein grafted with STP via N- and O-

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phosphate bonds. AVM@P-Zein also facilitated the anti-UV property and retained toxicity

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by protecting AVM with zein micelle. Overall, such nano-carrier system reduced the loss of

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AVM and improved the utilization of AVM, and thus will protect environment and human

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health to some extent. The formation mechanism of such AVM@P-Zein system is

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schematically shown in Scheme 1. hydrophobic drug

hydrophobic core

(AVM)

(Zein)

Zein pH=13

Phosphorylation water 134

hydrophilic shell (STP)

Zein disperse in water STP

water

Scheme 1. The illustration of AVM encapsulated in phosphorylated zein.

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MATERIALS AND METHODS

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Materials

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Zein (AR grade) was purchased from Shanghai Macklin Biochemical Co., Ltd

139

(Shanghai, China). Sodium tripolyphosphate (STP, Na5P3O10, 98%), sodium hydroxide

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(97%), hydrochloric acid (≥37%), ethanol anhydrous, acetone (≥99.5%), methanol (99.5%),

141

and acetate (99.5%) were procured from Damao Chemical Reagent Factory (Tianjin, China).

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Avermectin (AVM, ≥95%) was obtained from God Bull Pharmaceutical Chemical Co., Ltd

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(Wuhan, China) and used as received. AVM emulsifiable concentrate (technical grade) with

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5% mass ratio was obtained from Jiangsu Fengyuan Bioengineering Co., Ltd (Yancheng,

145

China). All chemicals were used directly without further purification. Deionized water was

146

used during all experiments.

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Preparation of phosphorylated zein

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The phosphorylated zein was prepared in the presence of STP (Scheme 2). In a typical

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procedure, a stock solution of zein was prepared by dissolving 1.0 g zein in 50 mL deionized

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water with pH 13 adjusted by NaOH in aim of dissolution completely by stirring under 30 ℃.

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Further, 1.0 g STP as the phosphorylation reagent was added to the above solution and then

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reacted by vigorous stirring at room temperature for 3 h. Then, the obtained solution was

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dialyzed in the presense of water for 48 h to remove impurities. Ultimately, the

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phosphorylated zein products were achieved by freeze-drying and designated as P-Zein-1, in

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which 1 indicated the mass ratio of zein to STP. Furthermore, other three samples with a

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variety mass ratio of zein to STP were also prepared and marked as P-Zein-2, P-Zein-3, and

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P-Zein-5, which represented the mass ratio as 2, 3, and 5, respectively. As depicted in

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Scheme 2, phosphate was combined with zein by N- and O- phosphate bonds after

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phosphorylation 42- 43. (a) NH H

Zein

N H

O PO O

STP

(b) OH

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Zein

O

O P O O

STP

161 162

Scheme 2. Synthetic routes of phosphorylated zein: (a) reaction between phosphate and N atom of zein, (b) reaction between phosphate and O atom of zein.

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Loading of AVM in phosphorylated zein

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The loading of AVM in phosphorylated zein was achieved by impregnation. In a typical

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process, 1.0 g AVM was added into 100 mL ethanol anhydrous, producing AVM solution

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concentration of 10 mg/mL. Then, 0.5 g phosphorylated zein was dissolved in 80 mL

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deionized water, sequentially, 10 mL AVM in ethanol solution (10 mg/mL) was added, and

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then the mixture was vigorously stirred for 1 h to fully dissolve the reactants to obtain

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AVM-loaded phosphorylated zein, which marked as AVM@P-Zein-1, AVM@P-Zein-2,

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AVM@P-Zein-3, and AVM@P-Zein-5, in accordance with mass ratio of zein to STP,

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respectively.

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Characterization of phosphorylated zein and AVM@phosphorylated zein

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Fourier transform infrared (FTIR) was carried out by a Spectrum 100 Fourier infrared

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spectrometer (PerkinElmer Inc., USA) using KBr pellet technique to characterize and

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compare the vibrational states of zein, phosphorylated zein, and AVM@P-Zein with 4 scans

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in the range of 4000-400 cm-1.

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The thermal stability of phosphorylated zein was analyzed by TGA 2 thermogravimetric

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analyzer (Mettler Toledo, Switzerland) under N2 atmosphere over the heating range of

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40-700 ℃ at a heating rate of 10 ℃/min.

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The motion transformation behavior of zein during phosphorylation was examined using

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a DSC-Q20 calorimeter (TA instruments, USA) equipped with a cooler by heating from 0 °C

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to 200 °C at a rate of 10 °C/min under a nitrogen atmosphere.

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The size distribution and zeta potential of phosphorylated zein were measured via a 90

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Plus PALS particle size and zeta potential analyzer (Bruker Corporation, USA), following

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diluting in Milli-Q water. The measurements were carried out at a scattering angle of 90° at

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25 ℃.

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The morphology, structure, and size of zein, phosphorylated zein, and AVM@P-Zein

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were taken by using EVO18 scan electron microscopy (Zeiss, Germany). For SEM

189

measurement, a droplet of sample solution was placed onto a clean surface, evaporating

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solvent at ambient temperature, and then coated with Au to minimize charging effects. The

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sample topography was obtained at an accelerating voltage of 15 kV.

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The phosphorylated zein’s wettability was determined from DAS 100 contact angle

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meter (Krüss GmbH, Germany). The system consisted of a flattened cucumber leaf as the

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solid substrate, and phosphorylated zein solution as the aqueous phase.

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The phosphorylation degree test of zein

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198 199

The degree of phosphorylation (Dp) was calculated according to the formula (1), where m0 is the original mass of zein, mP-Zein is the mass of phosphorylated zein after freeze-drying.

𝐷𝑝 =

𝑚𝑃 ― 𝑍𝑒𝑖𝑛 ― 𝑚0 𝑚𝑃 ― 𝑍𝑒𝑖𝑛

(1)

Encapsulation efficiency measurement

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The amount of AVM loaded into phosphorylated zein was quantified using the UV-2550

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ultraviolet-visible (UV-vis) spectroscopy (Shimadzu, Japan). The absorption at a wavelength

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of 245 nm, which is a characteristic peak for AVM, was selected in the analysis. Specifically,

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a given amount of AVM@P-Zein solution was centrifugated and then measured the

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concentration of AVM among upper transparent layer. The encapsulation efficiency (EE) of

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phosphorylated zein was achieved by the following formula (2), where mtotal (mg) is the total

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mass of AVM in solution, mfree AVM (mg) is the mass of unencapsulated AVM. EE (%)  ( mtotal AVM  m free AVM ) / mtotal AVM  100 %

(2)

Sustained release rate test

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The sustained release rates of AVM from phosphorylated zein were examined via a

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dialysis-based assay. Briefly, 5 mL of AVM@P-Zein solution was placed into a standard

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regenerated dialysis membrane (molecular weight cut-off 5000 Da) and dialyzed against 100

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mL 40 wt% ethanol in water at 25 ℃. The aliquots from the reservoir were collected at a

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variety of time and analyzed with a UV-2550 UV-Vis spectrometer (Shimadzu, Japan) to

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obtain the concentration of AVM at λ=245 nm. The cumulative release rate (Ri) of AVM was

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calculated according to the following formula (3), where ρi (mg/L) is the mass concentration

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of AVM among sample solution. All experiments were carried at least in triplicate.

217

218

{

𝜌𝑖 × 0.1/𝑚𝐴𝑉𝑀 (𝑖 = 1) 𝑖―1 𝑅𝑖 = 𝜌 × 0.1/𝑚 𝑖 𝐴𝑉𝑀 + ∑𝑖 = 1𝜌𝑖 × 0.001/𝑚𝐴𝑉𝑀 (𝑖 = 2, 3, 4...)

(3)

Anti-ultraviolet performance test

219

The anti-ultraviolet performance was carried out by placing AVM@P-Zein dispersing in

220

50 wt% ethanol solution under UV light (16 W Lamp) within a certain distance (20 cm).

221

Then, the aliquots from the solution were obtained at time intervals and measured absorbance

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with a UV-Vis spectrometer. Subsequently, the cumulative remaining rate (RR) was

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determined from the following formula (4), where A0 is the original absorbance of AVM in

224

solution, Ai is the absorbance for remaining AVM in solution after UV irradiation for a

225

certain time.

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227


RR 

Ai  100% A0

（4）

Leaf adhesion test

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The cucumber leaves were selected as the solid substrate, while same amounts of AVM

229

encapsulated in various phosphorylated zein solution, marked as AVM@P-Zein-1,

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AVM@P-Zein-2, AVM@P-Zein-3, and AVM@P-Zein-5, were chosen as liquid phase to

231

spray on fresh and clean cucumber leaves with the same size. Following that, the cucumber

232

leaves were dried at ambient condition and then washed with 100 mL deionized water. The

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sample adhesion ability on leaf was determined via residual rate of AVM as the formula (5),

234

which mainly calculated the AVM concentration in water, and where mAVM0 means the

235

original mass of AVM in solution, and mAVM1 is the remaining mass of AVM on leaves after

236

washing.

237

238

residual rate  (1 

m AVM 1 )  100% m AVM 0

(5)

Toxicity test of AVM@phosphorylated zein

239

The effect of phosphorylated zein on toxicity of AVM was evaluated via immersing

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cabbage leaves (size: 2 cm×2 cm) into corresponding various concentrations of

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AVM@P-Zein-1 solutions, drying at ambient conditions, and placing on a Petri dish (eleven

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insects for each) with the third instar larva of diamondback moth under three replicates. The

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Petri dishes with leaf and insects were cultured in the larval chamber. Afterwards, the

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mortality of treated insects was monitored at 24 h and 48 h after post-treatment and then

245

calculated in according to Abbott formula 44, where insects were considered dead if could not

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move or unable to move when prodding. The toxicity regression equations and lethal

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concentration of 50% (LC50) were fitted and obtained in light of probabilistic analysis. As a

248

control, AVM dispersing in ethanol solution was selected to treat on insects as well. Mortality

249

and corrected mortality were calculated.

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RESULTS AND DISCUSSION

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Structural characteristics

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Figures 1a-c compare typical morphology of zein, phosphorylated zein, and AVM

253

encapsulated with phosphorylated zein using SEM, respectively. As can be seen, raw zein

254

without any modification possessed spherical shapes with intimate connection topography, in

255

accordance with spiral structures proposed by Argos et al.

256

arranged in parallel and formed stable structures by hydrogen bonding

257

droplet size of zein obtained from SEM image was estimated to be 437±53 nm. Compared in

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Fig.1b, phosphorylated zein had much smaller spherical shapes with an approximately size of

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150±25 nm. In this case, the structured connection of zein was damaged via phosphorylation,

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which was presumably induced by the reaction between STP and amino group of zein,

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destroying the hydrogen bond to some extent. On the other hand, phosphate ions around zeins

262

can play a role in stabilizing dispersion due to electrostatic repulsion. Hence, phosphorylated

263

zein was not easy to aggregate and dispersed relative uniformly as depicted in Fig.1b.

264

Furthermore, structural morphology of AVM encapsulated in phosphorylated zein was shown

265

in Fig. 1c, from which can be obtained the average size of 216±52 nm. The particle size for

266

AVM encapsulated in P-Zein became larger than the cases of P-Zein under different

267

phosphorylation degrees as follows, which mainly was ascribed to the increase of the internal

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45,

in which multiple zeins were 46.

And the mean

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cavity. Besides, AVM encapsulated in phosphorylated zein produced the decreased sphericity

269

and poorer dispersity, which probably because ethanol as solvents for AVM decreased

270

group’s dielectric constant and charges on surface, and thus reducing the solution dispersity.

271

Intuitively, Fig.1d displays digital photos of zein and phosphorylated zein, in which zein can

272

not dissolve in water completely, but phosphorylation improved the solubility of zein in water

273

obviously due to the change of zein’s structure.

274

The particle size of phosphorylated zein in different degrees, marked as P-Zein-1,

275

P-Zein-2, P-Zein-3, and P-Zein-4, obtained from DLS was 152.53±10.25 nm, 180.88±8.33

276

nm, 192.41±9.76 nm, and 224.20±6.37 nm, respectively (as reported in Table 1), which

277

indicated that the size grew up with the additive increase of zein, and were also consistent

278

with the size measurements from SEM images.

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a-1

a

1 μm

b-1

b

1 μm

c

c-1

1 μm

d Zein

P-Zein

279 280 281 282 283 284

Figure 1. SEM images of (a) zein, (b) phosphorylated zein (P-Zein), (c) AVM@P-Zein, and (d) photograph of zein and P-Zein dispersing in aqueous solution captured by digital camera. And a-1, b-1, and c-1 referring to size distribution of zein, P-Zein, and AVM@P-Zein by counting more than twenty droplets and calculated by Image J from corresponding SEM images a, b, and c.

285

Table 1. Effect of phosphorylation degree on size and zeta potential of P-Zein. Sample

Particle size/nm

Zeta potential/mV

P-Zein-1

152.53±10.25

-66.47±6.29

P-Zein-2

180.88±8.33

-53.92±5.36

P-Zein-3

192.41±9.76

-48.64±8.37

P-Zein-5

224.20±6.37

-38.57±3.25

286

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Degree of phosphorylation and colloidal stability

288

As mentioned above, the mass ratio of zein to STP was chosen as 1, 2, 3, 5, which affect

289

the size of P-Zein. Besides, Figure 2 depicts the effect of amount of zein on phosphorylation

290

degree, which can be seen that the degree of phosphorylation (Dp) was 116.76±5.89 mg/g,

291

48.89±7.96 mg/g, 28.78±8.23 mg/g, and 13.96±4.15 mg/g, respectively. Namely, the degree

292

of phosphorylation gradually decreased as the amount increase of zein in the system, which

293

probably because the larger amount of zein, reacted with a certain amount of STP, produced

294

the less functional groups in the unit volume of reaction and thus the lower degree of

295

phosphorylation.

Phosphorylation degree (mg/g)

140 120 100 80 60 40 20 0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

The additive amount of zein (g)

296 297

Figure 2. Effect of additive amount of zein on phosphorylation degree.

298

Furthermore, the phosphorylated zein was stable against aggregation, which can be

299

claimed from relative higher zeta potential of P-Zein-1, P-Zein-2, P-Zein-3, and P-Zein-5.

300

The value for different degrees of phosphorylation was -66.47±6.29 mV, -53.92±5.36 mV,

301

-48.64±8.37 mV, and -38.57±3.25 mV, respectively (as described in Table 1), which also

302

present that absolute zeta potential became lower when phosphorylation degree reducing.

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This phenomenon can be presumably due to the less phosphoric groups and reduced negative

304

charge on surface, which also led to the decrease of free volume of particles, thus prone to

305

aggregation and increasing the particle size while lower degree of phosphorylation.

306

Thermal property analysis of phosphorylated zein

307

Compared TGA curves of zein and P-Zein in Figure 3a, the loss weight of water among

308

P-Zein below 100 ℃ became larger, mainly because holding capacity for water of protein

309

after phosphorylation was greater than that of raw Zein, also indicating that STP modified

310

zein successfully to improve its hydrophilic property. Besides, zein presented the onset

311

decomposed temperature at 258 ℃ with a decomposition peak at 327 ℃ in Fig.3b, while zein

312

after phosphorylation began to decompose at lower temperature (231 ℃) with a

313

decomposition peak at 299 ℃, which revealed that the thermal stability of phosphorylated

314

zein decreased. This phenomenon is likely to be ascribed to STP with small molecules

315

stepping into the interior of zein occurred reaction, resulting in a decrease of the internal

316

tightness of protein, thereby reducing its thermal stability.

(a)

(b) Derivative weight (%/℃ )

258 ºC

231 ºC

317 318 319

299 ºC

327 ºC

(ºC)

ºC

Figure 3. TGA (a) and DTG (b) curves of zein and P-Zein. To prove the transformation of zein molecular motion during phosphorylation, Figure 4 18


Page 19 of 39


320

depicted the DSC thermograms of zein and P-Zein under different phosphorylation degrees,

321

in which P-Zein 1-5 represented the reduction of phosphorylation degree as mentioned above.

322

Zein exhibited a broad endothermic peak at 86 °C contributed to relatively easy bound water

323

loss from hydrophobic molecules

324

approximate 155 °C. Intriguingly, the peak around 155 °C disappeared after phosphorylation,

325

indicating zein dispersed molecularly and interacted with STP strongly, and the peak

326

correspond to bound water was also not detected, presumably ascribed to the strong affinity

327

between P-Zein with water. Additionally, the endothermic peak among samples with various

328

phosphorylation degrees, indicative the glass transition temperature, gradually lift up from

329

105 °C to 121 °C as the decrease of phosphorylation degree, provided the evidence of

330

excellent compatibility between zein and STP and molecularly motion of P-Zein.

47,

155 C

86 C

Heat Flow

Zein P-Zein-1 P-Zein-2 P-Zein-3 P-Zein-5

and another tiny characteristic peak appeared at

105 C 111 C 118 C 121 C

0

331 332 333 334

25

50

75

100

125

150

175

200

Temperature (C)

Figure 4. DSC thermograms of zein and P-Zein with different phosphorylation degrees. Spectroscopic analysis into interaction in the system FTIR spectra was utilized to investigate the functional groups of zein, P-Zein, and

19



Page 20 of 39

335

AVM@P-Zein, as shown in Figure 5. In Zein spectrum, the peak at 3345 cm-1 was ascribed

336

to the stretching of N-H, the peak at 1663 cm-1 was associated with the stretching vibration of

337

-C=O for forming -CONH-, and the peak at 1536 cm-1 corresponded to the coupling of N-H

338

bending vibration and C-N stretching vibration among amide absorption band Ⅱ. Upon

339

phosphorylation using STP regarded as P-Zein, it occurred the peak ascribed to P-O

340

stretching vibration at 1122 cm-1 and the peak contributed to the asymmetric stretching

341

vibration of phosphate at 1013 cm-1, which proved STP grafted with Zein successfully.

342

Comparatively, the FTIR spectrum for AVM encapsulated in P-Zein, marked as

343

AVM@P-Zein, appeared two new peaks at 1450 cm-1 and 1100 cm-1 corresponded to the

344

deformation vibration of C-O-H for AVM (IV) and the stretching vibration of C-O-C for

345

AVM (IV) hybridization. Meanwhile, the characteristic peaks of P-Zein in AVM@P-Zein

346

system can not be seen obvious changes, which indicated that P-Zein encapsulated AVM

347

mainly via physical interaction.

I

II

1663 1536 1122 1013

1100 1456

4000

3500

3000

2500

2000

1500

1000

III

IV

500

-1

348 349

Wavenumber (cm )

Figure 5. FTIR spectra of zein (I), P-Zein (II), AVM@P-Zein (III) and AVM (IV).

20


Page 21 of 39

350


Encapsulation behavior of P-Zein for AVM

351

AVM loaded behavior inside P-Zein was investigated and the amount of encapsulated

352

AVM in P-Zein was quantified by UV-Vis analysis. To this end, the effect of parameters, i.e.

353

phosphorylation degree and pH, were analyzed as follows.

354

Effect of phosphorylation degree of P-Zein on encapsulation efficiency

355

Table 2 also summarized the AVM encapsulation efficiency values of a variety of

356

P-Zein with different phosphorylation degrees under the condition of pH 7. Correspondingly,

357

the encapsulation efficiency of AVM@P-Zein-1 reached 81.52±2.31%, relatively higher than

358

other three cases of AVM@P-Zein. Yet, the encapsulation efficiency gradually decreases

359

with the phosphorylation degree becoming lower. In detail, when the degree of

360

phosphorylation decreased from 116.76 mg/g (regarded as P-Zein-1) to 28.78 mg/g (regarded

361

as P-Zein-3), the corresponding encapsulation efficiency reduced from 81.52±2.31% to

362

80.96±3.76%. This may be explained that the loading amount of P-Zein-3 almost became

363

saturated, hence, the continued increase of phosphorylation degree made the change of

364

encapsulation efficiency slight, only rising the negative charge on surface and decreasing the

365

particle size as indicated in Table 2. Furthermore, the encapsulation efficiency decreased

366

obviously from 80.96±3.76% for P-Zein-3 to 67.92±2.87% for P-Zein-5 with

367

phosphorylation degree 13.96 mg/g, which probably because the increase of particle size led

368

to the reduction of specific area and thus resulted in trapping rate of P-Zein as carrier for

369

AVM decreasing. In addition, lower surface electronegative charge density for P-Zein-5

370

could easily lead to agglomeration of AVM-loaded particles, destroying the structure of

21



Page 22 of 39

371

particles and then resulting in the release of AVM, thus leading to the reduction of

372

encapsulation efficiency.

373 374

Table 2. Effect of phosphorylation degree and pH on size, zeta potential, and encapsulation efficiency of AVM@P-Zein.

Sample AVM@P-Zein1

AVM@P-Zein2 AVM@P-Zein3 AVM@P-Zein5

Zeta

pH

Particle size/nm

7

174.09±7.31

-60.34±2.11

81.52±2.31

3

Precipitate

--

Precipitate

5

Precipitate

--

Precipitate

7

199.58±5.25

-49.82±2.54

81.05±1.55

9

156.77±6.91

-54.06±1.79

81.23±1.75

7

215.63±10.89

-44.93±2.31

80.96±3.76

7

278.47±5.62

-30.64±1.25

67.92±2.87

potential/mV

EE/%

375 376

Effect of pH on encapsulation efficiency

377

AVM@P-Zein-2 was used as criteria to study the effect of pH on encapsulation

378

efficiency, as depicted in Table 2. As observed during experiments, AVM appeared

379

precipitates in P-Zein-2 solution when adjusted pH to 3 and 5, which can be interpreted as the

380

protonation of phosphate in P-Zein-2 to form phosphoric acid under acidic conditions due to

381

the isoelectric point of zein of 6.5 48, thus inducing the reduction of electronegative charge on

382

the surface of P-Zein-2 and repulsive force among AVM@P-Zein-2, ultimately, favoring the

383

formation of aggregation and precipitation. Nevertheless, the encapsulation efficiency values

384

(81.23±1.75) under weak base conditions was close to the case of neutral condition (pH=7)

22


Page 23 of 39


385

with encapsulation efficiency (81.05±2.03), which means both of neural and base conditions

386

possessed negative phosphate ions beneficial to the dispersion of AVM in P-Zein solution.

387

Sustained release behavior of AVM from P-Zein

388

Effect of phosphorylation degree of P-Zein on sustained release rate

389

To evaluate the sustained release property of AVM@P-Zein, firstly, we carried out the

390

experiments using different phosphorylation degree samples. As displayed in Figure 6, the

391

trend for sustained release of AVM among various AVM@P-Zeins only had slight

392

differences. In initial stage of 24 h, AVM released from AVM@P-Zeins was very rapid due

393

to the presence of unencapsulated AVM resulted in “burst release”. Continually, the release

394

rate of AVM gradually became calm after 98 h sustained releasing. Ultimately, the sustained

395

release rate for AVM@P-Zein-1, AVM@P-Zein-2, AVM@P-Zein-3, and AVM@P-Zein-5

396

was 45.33±1.13%, 44.79±1.87%, 42.91±1.59%, and 45.71±1.85%, respectively at sustained

397

release time 337 h. The sustained-release test was only carried out for 337 h, which still

398

remained approximately 55% AVM encapsulated in P-Zein ascribed to its excellent

399

protection. However, ultimately, this part of AVM residue would be release totally due to the

400

damage of P-Zein for a longer time due to P-Zein’s biodegradable property. This

401

phenomenon is suitable for some certain crop or plant growth conditions, such as it needs

402

slow-release of pesticides in initial growth stage, then with longer time of growth, it required

403

a large amount of pesticides to protect products from the damage of P-Zein.

23



Page 24 of 39

50

Cumulative release rate (%)

45 40

AVM@P-Zein-1 AVM@P-Zein-2 AVM@P-Zein-3 AVM@P-Zein-5

35 30 25 20 15 10 5 0

0

50

100

150

250

300

350

Time (h)

404 405 406

200

Figure 6. The cumulative release rates for different AVM@P-Zein formulations as a function of releasing time.

407

Effect of pH on sustained release rate

408

The comparison of sustained release rates under different pH conditions, i.e. pH 3, 5, 7,

409

and 9 for AVM@P-Zein-3, was reported in Figure 7. As observed, the release trend of AVM

410

presented exponentially for the initial 25 h due to the unencapsulated AVM permeated

411

rapidly from dialysis bag, and subsequently the release rate became slow which beneficial for

412

prolonging the duration of AVM and thus reducing the required amount of pesticides.

413

Comprehensively, the sustained release rates under acidic conditions (pH 3 and 5) was

414

greater approximately 6.03% than those under base (pH 9) and neutral (pH 7) conditions.

415

Comparatively, the most effective means to sustained release was under neutral conditions

416

applicable to actual situation for utilizing pesticides. This phenomenon was ascribed to the

417

formation of precipitate for AVM@P-Zein under acidic conditions with poor dispersity owe

418

to protonation as aforementioned and hence accelerating the releasing of core AVM obtained

419

the utmost cumulative release rate under acidic cases. Furthermore, isoelectric point of zein is

24


Page 25 of 39


48,

420

6.5 as literature reported

while P-Zein via phosphorylation possessed less amino groups

421

and more quantity of phosphate ions, resulting in the reduction of isoelectric point. Thereby,

422

it was presumably approached to isoelectric point at pH of 3 and 5, favoring the aggregation

423

of AVM@P-Zein and promoting the pesticide release. On the other hand, it deviated more

424

from isoelectric point under pH 9 and AVM@P-Zein also carried more negative charges with

425

better stability as aforementioned to generate electrostatic repulsion, restricting the interior

426

AVM to escape at pH of 9. Besides, the interior cavity of P-Zein became extending and

427

interior electrostatic interaction weakened under neutral conditions due to deprotonation,

428

evidenced from Table 1 and 2, which caused the reduction of pesticide escaping resistance

429

and speed up pesticide release. Overall, the minimum cumulative release rate appeared at pH

430

of 7.

50

Cumulative release rate (%)

45 40

pH=3 pH=5 pH=7 pH=9

35 30 25 20 15 10 5 0

0

50

434

150

200

250

300

350

Time (h)

431 432 433

100

Figure 7. Effects of pH on sustained-release performance of AVM from P-Zein. Release kinetics mechanism

25



Page 26 of 39

435

In order to further investigate the mechanism of release kinetics of AVM from P-Zein,

436

we selected several kinetic models to analyze the aforementioned sustained release data under

437

a variety range of pH conditions, including zero-order 49, first-order 50, Korsmeyer-Peppas 51,

438

Elovich

439

which can be concluded that the sustained release kinetic curves fitted to first-order

440

and Elovich model better with relatively higher regression coefficient R2. The

441

first-order model implied the release of AVM was related to drug concentration,

442

wherein the exponent index a1 indicated the release rate and constant a0 showed

443

maximum cumulative release capacity, from the obtained results we can demonstrated

444

that the release rate was more rapid, achieving maximum cumulative amount under pH

445

of 5. Fitting to Elovich model with higher R2 implied the diffusion process was a

446

heterogeneous diffusion from carrier with swelling of carrier, controlled by interaction

447

rate and diffusion factor 54.

448

52,

and Weibull model

53.

The fitted results were reported as Table 3, from

Table 3. Fitting results for the data of sustained release of AVM at different pH values. Kinetic model

Formula

First-order

Q = a0(1-exp(-a1t))

Zero-order

Q = a0t

Korsmeyer-Peppas

Q = a0ta1

Elovich

Q=(1/a0)*(ln(a0*a1)+ln(t))

26

pH 3 5 7 9 3 5 7 9 3 5 7 9 3 5 7 9


a0 a1 45.6241 0.2082 46.2845 0.1445 40.7858 0.1978 41.4692 0.1563 0.2112 3.8962 3.4883 3.5332 17.3988 0.2187 15.2702 0.2434 13.9600 0.2442 13.9642 0.2426 0.1686 145.7184 0.1597 90.7225 0.1822 100.5032 0.1775 85.8240

R2 0.9237 0.9354 0.9588 0.9275 0.4528 0.7834 0.7547 0.7872 0.9033 0.9192 0.8864 0.9328 0.9293 0.9202 0.9089 0.9457

Page 27 of 39


3 5 7 9

449 450

0.2208 0.2434 Weibull Q= 0.2184 0.2334 Note: In release formula, Q represented cumulative release rate (%), t (h), and a0 and a1 defined as kinetics constants.

451

Foliage wettability and adhesion ability

1-exp(-(ta0)/a1)

4.4769 0.8858 5.0719 0.8824 5.2286 0.8412 5.5562 0.9025 referred release time

452

To evaluate the wetting characteristics of phosphorylated zein and AVM encapsulated in

453

P-Zein, static contact angle measurements were conduct on cucumber leaves and the photos

454

were displayed in Figure 8. As a control, the contact angle of pure water on cucumber leaf

455

was 90.44±0.35° (Fig.8a), the values above 90° demonstrating the surface of cucumber leaf

456

hydrophobic. Meanwhile, the contact angle of AVM in ethanol solution on leaf was

457

approximately 27.97±1.75° (Fig.8b), indicating AVM dissolve in ethanol can wet the

458

cucumber leaf. On the other hand, P-Zein solution can also wet the cucumber leaf to some

459

extent, which can be claimed from the contact angle of P-Zein on leaf 54.34±0.98° (Fig.8c).

460

This also suggested that wettability of P-Zein was better than water but weaker than AVM in

461

ethanol solution, which can be interpreted as follows: ethanol as organic solvents can wet

462

hydrophobic surface, while the solvent for P-Zein solution was water, thus weaken the

463

wettability on hydrophobic surface compared to ethanol. However, P-Zein can reduce the

464

surface tension of water, hence, improved the wettability compared with water. Once

465

encapsulated AVM in P-Zein, the contact angle between AVM@P-Zein and cucumber leaf

466

reduced to 43.39±1.23° (Fig.8d), which larger than that of AVM in ethanol solution but

467

smaller than that of P-Zein solution due to the introduce of a small amount of ethanol as

468

dispersing media when dissolving AVM powder.

27



a

b

c

d

469 470 471

Figure 8. Contact angle snapshots of (a) water, (b) AVM in ethanol solution, (c) P-Zein, and (d) AVM@P-Zein on cucumber leaves.

472

To confirm the adhesion ability of AVM@P-Zein, we tried to immerse cucumber leaves

473

sprayed with AVM@P-Zein-1 and AVM@P-Zein-5 samples into water to erode, meanwhile,

474

we selected cucumber leaves with AVM alone dispersing in ethanol as a control. As observed

475

from Figure 9, the residual rate of AVM from P-Zein-1 and P-Zein-5 with different

476

phosphorylation degree on cucumber leaves after water eroding was 39.10±1.12% and

477

37.65±0.87%, respectively, both of them higher than the value of bare AVM dispersing in

478

ethanol 33.49±0.55%. The results among the three samples implied that encapsulation by

479

P-Zein improved the adhesion ability of AVM on cucumber leaves, which presumably

480

contributed to zein favored the film formation of AVM@P-Zein on cucumber leaves 55, and

481

thus was beneficial for adhering on hydrophobic surface. Nevertheless, phosphorylation of

482

Zein enhanced the hydrophilicity significantly as mentioned above, leading to a certain

483

amount of Zein wrapped on AVM@P-Zein preferred to dissolve in water. Considering these

28


Page 28 of 39

Page 29 of 39


484

two factors, the intensification of adhesion ability of AVM@P-Zein was not much obviously

485

compared to AVM dispersing in ethanol.

33.49±0.55

39.10±1.12

35.17±0.97

36.52±0.67

37.65±0.87

486 487 488 489

Figure 9. AVM residual amount on cucumber leaves after eroding with water, in which A, B, C, D, and E referred to AVM dispersing in ethanol solution, AVM@P-Zein-1, AVM@P-Zein-2, AVM@P-Zein-3, and AVM@P-Zein-5, respectively.

490

Anti-UV performance of AVM@P-Zein

491

The relationship between the residual rate of AVM using the cumulative remaining rate

492

(RR) as criteria and the irradiation time under UV light was depicted as Figure 10. Initially,

493

AVM without encapsulation degraded fast under UV light. Specifically, the cumulative

494

remaining rate became 86.71±2.11% after 1 h irradiation. Continually irradiated under UV

495

light for 7 h, the degradation of AVM turned more rapid with the remaining rate

496

51.86±1.32%. Furthermore, the cumulative remaining rate of AVM in ethanol solution only

497

retained 37.70±1.56% at irradiation time for 43 h, from which obtained the half-life for bare

498

AVM was 11 h. It also can be seen that anti-UV performance of commercial AVM

499

emulsifiable oil equaled to AVM in ethanol solution under Ultraviolet irradiation, which

29



Page 30 of 39

500

indicated that the application of AVM emulsifiable oil in agriculture could not improve the

501

stability of active ingredient much, even would pollute the environment and destroy the

502

sustainable development of agriculture from usage of a large amount of organic solvents.

503

However noticeably, the case of AVM encapsulated in P-Zein made the degradation speed

504

become much slower. In detail, as for AVM@P-Zein-1, the remaining rate of AVM was

505

92.85±0.75% for 1 h irradiation, and then this value reduced to 74.75±1.23% for irradiating

506

for 7 h. When prolonging the irradiation time to 43 h, the cumulative remaining rate of AVM

507

inside P-Zein media still maintained 65.13±2.11%. As compared above, AVM encapsulated

508

in P-Zein enhanced the stability of bare AVM against UV irradiation, which was ascribed to

509

P-Zein as carriers and physical barriers restricted the AVM exposed directly to UV light, and

510

further weakening the intensity of UV light.

Cumulative remaining rate (%)

100 90 80 70 60 50 40

20 10 0

513 514

0

5

10

15

20

25

30

35

40

45

50

55

60

Irradiation time (h)

511 512

5% AVM emulsifiable concentrate (commercial) AVM in ethanol solution AVM@P-Zein-1 AVM@P-Zein-2 AVM@P-Zein-3 AVM@P-Zein-5

30

Figure 10. Remaining AVM amount after UV light irradiation. Toxicity analysis For the sake of assessing the toxicity of AVM encapsulated in P-Zein, LC50 and 95%

30


Page 31 of 39


515

confidence limit were used as criteria of toxicity. Table 4 compared the toxicity results via

516

probability analysis of AVM@P-Zein as well as AVM dispersing in ethanol solution and

517

AVM emulsifiable concentrate as references. In detail, the LC50 values achieved from

518

corresponding toxicity regression equations for AVM in ethanol solution, AVM emulsifiable

519

concentrate, and AVM@P-Zein were 26.22, 27.04, and 41.73 mg/L, respectively. The

520

application of P-Zein encapsulating AVM induced the growth of LC50, indicative of the

521

protection and slow-release of AVM by P-Zein owing to interfacial barrier layer, and

522

enhancing its duration and utilization. Moreover, regarding the 95% confidence limit in these

523

three cases experiencing some situation, a part of this value for AVM@P-Zein located in the

524

range for the case of AVM using ethanol as dispersing media and AVM emulsifiable

525

concentrate, suggesting that AVM@P-Zein had a similar toxicity effect with AVM dispersing

526

in ethanol solution and commercial AVM emulsifiable concentrate, and also providing a

527

strong evidence for no significant change of toxicity after encapsulation and protection of

528

AVM by P-Zein.

529 530

Table 4. Results of toxicity analysis for AVM dispersing in ethanol solution, commercial AVM emulsifiable concentrate, and AVM@P-Zein.

Sample AVM dispersing in ethanol solution AVM emulsifiable concentrate AVM@P-Zein

Toxicity regression equation

LC50（mg/L）

95% confidence limit

R2

Y=1.9300+2.1642X

26.22

18.87~36.43

0.9060

Y=2.4407+1.7872X

27.04

19.34~37.81

0.9628

Y=2.1973+1.7296X

41.73

30.94~56.28

0.9718

531 532

In this study, a water-soluble phosphorylated zein with AVM payload was prepared and

31



533

investigated as aqueous-based nano-carrier system to improve the foliage wettability and

534

utilization of pesticides. Such nano-carrier system was fabricated using the modification of

535

zein by sodium triphosphate and encapsulation of AVM by hydrophobic-hydrophobic

536

interaction, and had fairly spherical structures with average particle sizes in the range of

537

174-278 nm. UV-vis measurements manifested that phosphorylated zein encapsulated AVM

538

durably and effectively with encapsulation efficiency of 81.52%, having an excellent stability

539

evidenced from relatively high absolute zeta potential values. The release of AVM can be

540

induced from such nano-carriers via the application of swelling in ethanol solution and with

541

pH-sensitivity owe to the phosphate protonation and phosphoric acid deprotonation fitting to

542

Elovich model, augmented the release rate under acidic conditions (48.93%). Moreover, we

543

also demonstrated that AVM@P-Zein could enhance anti-UV performance significantly and

544

extend the half-life of AVM effectively. Furthermore, AVM@P-Zein had a strong potential

545

in effectively improving wettability on hydrophobic leaf with contact angle of 43.39° and

546

providing an effective approach to avoid the pesticides loss by strengthening the adhesion

547

ability on leaf with residual rate of 39.10% after eroding. More significantly, AVM@P-Zein

548

retained the toxicity level similar with bare AVM. Overall, such aqueous-based nano-carrier

549

can be considered as pesticide-loaded nanocapsules that protecting pesticide from loss and

550

photodegradation and thus improving the utilization effectively.

551 552

AUTHOR INFORMATION

553

Corresponding Authors

554

*Telephone: 86-20-89003114. E-mail: [email protected] (Hongjun Zhou), School of

32


Page 32 of 39

Page 33 of 39


555

Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering,

556

Guangzhou, Guangdong, 510225, P. R. China.

557

*Telephone: 86-20-89003114. E-mail: [email protected] (Xinhua Zhou), School of

558

Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering,

559

Guangzhou, Guangdong, 510225, P. R. China.

560

Notes

561

The authors declare no competing financial interest.

562

ACKNOWLEDGEMENT

563

The authors gratefully appreciate the financial support from National Natural Science

564

Foundation of China (Grant 21606262), Natural Science Foundation of Guangdong Province

565

(Grant No. 2016A030313375, 2017A030311003), Science and Technology Program of

566

Guangzhou, China (Grant No. 201903010011).

567

REFERENCES

568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

(1) Zhao, X.; Cui, H.; Wang, Y.; Sun, C.; Cui, B.; Zeng, Z., Development strategies and prospects of nano-based smart pesticide formulation. Journal of Agricultural and Food Chemistry 2017, 66 (26), 6504-6512. (2) Roy, A.; Singh, S. K.; Bajpai, J.; Bajpai, A. K., Controlled pesticide release from biodegradable polymers. Central European Journal of Chemistry 2014, 12 (4), 453-469. (3) Liu, B.; Wang, Y.; Yang, F.; Wang, X.; Shen, H.; Cui, H.; Wu, D., Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules. Colloids and Surfaces B: Biointerfaces 2016, 144, 38-45. (4) Quaglia, F.; Barbato, F.; De Rosa, G.; Granata, E.; Miro, A.; La Rotonda, M. I., Reduction of the environmental impact of pesticides: waxy microspheres encapsulating the insecticide carbaryl. Journal of Agricultural and Food Chemistry 2001, 49 (10), 4808-4812. (5) Kumar, S.; Chauhan, N.; Gopal, M.; Kumar, R.; Dilbaghi, N., Development and evaluation of alginate–chitosan nanocapsules for controlled release of acetamiprid. International Journal of Biological Macromolecules 2015, 81, 631-637. (6) Yao, J.; Cui, B.; Zhao, X.; Wang, Y.; Zeng, Z.; Sun, C.; Yang, D.; Liu, G.; Gao, J.; Cui, H., Preparation, characterization, and evaluation of azoxystrobin nanosuspension produced by wet media milling. Applied Nanoscience 2018, 8 (3), 297-307.

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(7) Yegin, Y.; Yegin, C.; Oh, J. K.; Orr, A.; Zhang, M.; Nagabandi, N.; Severin, T.; Villareal, T. A.; Sari, M. M.; Castillo, A., Ecotoxic effects of paclitaxel-loaded nanotherapeutics on freshwater algae, Raphidocelis subcapitata and Chlamydomonas reinhardtii. Environmental Science: Nano 2017, 4 (5), 1077-1085. (8) Li, Y.; Zhou, M.; Pang, Y.; Qiu, X., Lignin-based microsphere: preparation and performance on encapsulating the pesticide avermectin. ACS Sustainable Chemistry & Engineering 2017, 5 (4), 3321-3328. (9) Ezhilarasi, P.; Karthik, P.; Chhanwal, N.; Anandharamakrishnan, C., Nanoencapsulation techniques for food bioactive components: a review. Food and Bioprocess Technology 2013, 6 (3), 628-647. (10) George, M.; Abraham, T. E., Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. Journal of Controlled Release 2006, 114 (1), 1-14. (11) Chen, H.; Lin, Y.; Zhou, H.; Zhou, X.; Gong, S.; Xu, H., Synthesis, characterization of chlorpyrifos/copper(II) schiff base mesoporous silica with pH-sensitivity for pesticide sustained released. Journal of Agricultural & Food Chemistry 2016, 64 (43), 8095–8102. (12) Kumari, A.; Yadav, S. K.; Yadav, S. C., Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces B: Biointerfaces 2010, 75 (1), 1-18. (13) Mora-Huertas, C.; Fessi, H.; Elaissari, A., Polymer-based nanocapsules for drug delivery. International Journal of Pharmaceutics 2010, 385 (1-2), 113-142. (14) Gomollón-Bel, F., Ten Chemical Innovations That Will Change Our World: IUPAC identifies emerging technologies in Chemistry with potential to make our planet more sustainable. In Chemistry International, 2019; Vol. 41, p 12. (15) He, Q.; Shi, J., Mesoporous silica nanoparticle based nano drug delivery systems: synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility. Journal of Materials Chemistry 2011, 21 (16), 5845-5855. (16) Lin, G.; Zhou, H.; Lian, J.; Chen, H.; Xu, H.; Zhou, X., Preparation of pH-responsive avermectin/feather keratin-hyaluronic acid with anti-UV and sustained-release properties. Colloids and Surfaces B: Biointerfaces 2019, 175, 291-299. (17) Teng, Z.; Luo, Y.; Wang, Q., Nanoparticles synthesized from soy protein: preparation, characterization, and application for nutraceutical encapsulation. Journal of Agricultural and Food Chemistry 2012, 60 (10), 2712-2720. (18) Park, M.; Shin, H. K.; Kim, B.-S.; Kim, M. J.; Kim, I.-S.; Park, B.-Y.; Kim, H.-Y., Effect of discarded keratin-based biocomposite hydrogels on the wound healing process in vivo. Materials Science and Engineering: C 2015, 55, 88-94. (19) Tyler, B.; Gullotti, D.; Mangraviti, A.; Utsuki, T.; Brem, H., Polylactic acid (PLA) controlled delivery carriers for biomedical applications. Advanced Drug Delivery Reviews 2016, 107, 163-175. (20) Mondal, D.; Griffith, M.; Venkatraman, S. S., Polycaprolactone-based biomaterials for tissue engineering and drug delivery: Current scenario and challenges. International Journal of Polymeric Materials and Polymeric Biomaterials 2016, 65 (5), 255-265. (21) Sahoo, S.; Sasmal, A.; Nanda, R.; Phani, A.; Nayak, P., Synthesis of chitosan– polycaprolactone blend for control delivery of ofloxacin drug. Carbohydrate Polymers 2010, 79 (1), 106-113.

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Page 34 of 39

Page 35 of 39

629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672


(22) Campos, E. V. R.; De Oliveira, J. L.; Fraceto, L. F.; Singh, B., Polysaccharides as safer release systems for agrochemicals. Agronomy for Sustainable Development 2015, 35 (1), 47-66. (23) Kacsó, T.; Neaga, I. O.; Erincz, A.; Astete, C. E.; Sabliov, C. M.; Oprean, R.; Bodoki, E., Perspectives in the design of zein-based polymeric delivery systems with programmed wear down for sustainable agricultural applications. Polymer Degradation and Stability 2018, 155, 130-135. (24) Patel, A. R.; Velikov, K. P., Zein as a source of functional colloidal nano-and microstructures. Current Opinion in Colloid & Interface Science 2014, 19 (5), 450-458. (25) Li, H.; Wang, D.; Liu, C.; Zhu, J.; Fan, M.; Sun, X.; Wang, T.; Xu, Y.; Cao, Y., Fabrication of stable zein nanoparticles coated with soluble soybean polysaccharide for encapsulation of quercetin. Food Hydrocolloids 2019, 87, 342-351. (26) Dehcheshmeh, M. A.; Fathi, M., Production of core-shell nanofibers from zein and tragacanth for encapsulation of saffron extract. International Journal of Biological Macromolecules 2019, 122, 272-279. (27) Glusac, J.; Davidesko-Vardi, I.; Isaschar-Ovdat, S.; Kukavica, B.; Fishman, A., Gel-like emulsions stabilized by tyrosinase-crosslinked potato and zein proteins. Food Hydrocolloids 2018, 82, 53-63. (28) Paliwal, R.; Palakurthi, S., Zein in controlled drug delivery and tissue engineering. Journal of Controlled Release 2014, 189, 108-122. (29) Soltani, S.; Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oil-loaded zein nanoparticles. Food Hydrocolloids 2015, 43, 664-669. (30) Oliveira, J. L. d.; Campos, E. n. V.; Pereira, A. E.; Pasquoto, T.; Lima, R.; Grillo, R.; Andrade, D. J. d.; Santos, F. A. d.; Fraceto, L. F., Zein nanoparticles as eco-friendly carrier systems for botanical repellents aiming sustainable agriculture. Journal of Agricultural and Food Chemistry 2018, 66 (6), 1330-1340. (31) Zou, Y.; van Baalen, C.; Yang, X.; Scholten, E., Tuning hydrophobicity of zein nanoparticles to control rheological behavior of Pickering emulsions. Food hydrocolloids 2018, 80, 130-140. (32) Patel, A. R.; Bouwens, E. C.; Velikov, K. P., Sodium caseinate stabilized zein colloidal particles. Journal of Agricultural and Food Chemistry 2010, 58 (23), 12497-12503. (33) Krupa, A.; Preethi, G.; Srinivasan, N., Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases. Journal of Molecular Biology 2004, 339 (5), 1025-1039. (34) Nayak, S.; Arora, S.; Sindhu, J.; Sangwan, R., Effect of chemical phosphorylation on solubility of buffalo milk proteins. International Dairy Journal 2006, 16 (3), 268-273. (35) Zhang, K.; Li, Y.; Ren, Y., Research on the phosphorylation of soy protein isolate with sodium tripoly phosphate. Journal of Food Engineering 2007, 79 (4), 1233-1237. (36) Xiong, Z.; Zhang, M.; Ma, M., Emulsifying properties of ovalbumin: Improvement and mechanism by phosphorylation in the presence of sodium tripolyphosphate. Food Hydrocolloids 2016, 60, 29-37. (37) Enomoto, H.; Li, C. P.; Morizane, K.; Ibrahim, H.; Sugimoto, Y.; Ohki, S.; Ohtomo, H.; Aoki, T., Improvement of Functional Properties of Bovine Serum Albumin through Phosphorylation by Dry‐Heating in the Presence of Pyrophosphate. Journal of Food Science

35



673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716

2008, 73 (2), C84-C91. (38) Matheis, G., Phosphorylation of food proteins with phosphorus oxychloride—Improvement of functional and nutritional properties: A review. Food Chemistry 1991, 39 (1), 13-26. (39) Makara, A.; Smol, M.; Kulczycka, J.; Kowalski, Z., Technological, environmental and economic assessment of sodium tripolyphosphate production–a case study. Journal of Cleaner Production 2016, 133, 243-251. (40) Srivastava, G.; Walke, S.; Dhavale, D.; Gade, W.; Doshi, J.; Kumar, R.; Ravetkar, S.; Doshi, P., Tartrate/tripolyphosphate as co-crosslinker for water soluble chitosan used in protein antigens encapsulation. International Journal of Biological Macromolecules 2016, 91, 381-393. (41) Wang, Y.-R.; Zhang, B.; Fan, J.-L.; Yang, Q.; Chen, H.-Q., Effects of sodium tripolyphosphate modification on the structural, functional, and rheological properties of rice glutelin. Food Chemistry 2019, 281, 18-27. (42) Vojdani F.; Whitaker J. R.; Phosphorylation of proteins and their functional and structural properties. Macromolecular Interactions in Food Technology 1996, 210-229. (43) Matheis G.; Phosphorylation of food proteins with phosphorus oxychloride-improvement of functional and nutritional properties: a review. Food Chemistry 1991, 39(1), 13-26. (44) Fleming, R.; Retnakaran, A., Evaluating single treatment data using Abbott’s formula with reference to insecticides. Journal of Economic Entomology 1985, 78 (6), 1179-1181. (45) Argos, P.; Pedersen, K.; Marks, M. D.; Larkins, B. A., A structural model for maize zein proteins. Journal of Biological Chemistry 1982, 257 (17), 9984-9990. (46) Wang, Y.; Padua, G. W., Nanoscale characterization of zein self-assembly. Langmuir 2012, 28 (5), 2429-2435. (47) Luo, Y.; Teng, Z.; Wang, Q., Development of zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3. Journal of Agricultural and Food Chemistry 2012, 60 (3), 836-43. (48) Ji, N.; Hong, Y.; Gu, Z.; Cheng, L.; Li, Z.; Li, C., Preparation and characterization of insulin-loaded zein/carboxymethylated short-chain amylose complex nanoparticles. Journal of Agricultural and Food Chemistry 2018, 66 (35), 9335-9343. (49) Hsieh, D. S.; Rhine, W. D.; Langer, R., Zero-order controlled-release polymer matrices for micro-and macromolecules. Journal of Pharmaceutical Sciences 1983, 72 (1), 17-22. (50) Singhvi, G.; Singh, M., In-vitro drug release characterization models. International Journal of Pharmaceutical Studies and Research 2011, 2 (1), 77-84. (51) Basak, S.; Reddy, B. J.; Mani, K. L., Formulation and release behaviour of sustained release ambroxol hydrochloride HPMC matrix tablet. Indian Journal of Pharmaceutical Sciences 2006, 68 (5). (52) Chien, S.; Clayton, W., Application of Elovich equation to the kinetics of phosphate release and sorption in soils 1. Soil Science Society of America Journal 1980, 44 (2), 265-268. (53) Papadopoulou, V.; Kosmidis, K.; Vlachou, M.; Macheras, P., On the use of the Weibull function for the discernment of drug release mechanisms. International Journal of Pharmaceutics 2006, 309 (1-2), 44-50.

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(54) Cazalbou, S.; Bertrand, G.; Drouet, C., Tetracycline-loaded biomimetic apatite: an adsorption study. The Journal of Physical Chemistry B 2015, 119 (7), 3014-3024. (55) Liu, Z.; Cao, X.; Ren, S.; Wang, J.; Zhang, H., Physicochemical characterization of a zein prepared using a novel aqueous extraction technology and tensile properties of the zein film. Industrial Crops and Products 2019, 130, 57-62.

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722

Captions of figures

723

Scheme 1. The illustration of AVM encapsulated in phosphorylated zein.

724 725

Scheme 2. Synthetic routes of phosphorylated zein: (a) reaction between phosphate and N atom of zein, (b) reaction between phosphate and O atom of zein.

726 727 728 729 730

Figure 1. SEM images of (a) zein, (b) phosphorylated zein (P-Zein), (c) AVM@P-Zein, and (d) photograph of zein and P-Zein dispersing in aqueous solution captured by digital camera. And a-1, b-1, and c-1 referring to size distribution of zein, P-Zein, and AVM@P-Zein by counting more than twenty droplets and calculated by Image J from corresponding SEM images a, b, and c.

731

Figure 2. Effect of additive amount of zein on phosphorylation degree.

732

Figure 3. TGA (a) and DTG (b) curves of zein and P-Zein.

733

Figure 4. DSC thermograms of zein and P-Zein with different phosphorylation degrees.

734

Figure 5. FTIR spectra of zein (I), P-Zein (II), AVM@P-Zein (III) and AVM (IV).

735 736

Figure 6. The cumulative release rates for different AVM@P-Zein formulations as a function of releasing time.

737

Figure 7. Effects of pH on sustained-release performance of AVM from P-Zein.

738 739

Figure 8. Contact angle snapshots of (a) water, (b) AVM in ethanol solution, (c) P-Zein, and (d) AVM@P-Zein on cucumber leaves.

740 741 742

Figure 9. AVM residual amount on cucumber leaves after eroding with water, in which A, B, C, D, and E referred to AVM dispersing in ethanol solution, AVM@P-Zein-1, AVM@P-Zein-2, AVM@P-Zein-3, and AVM@P-Zein-5, respectively.

743

Figure 10. Remaining AVM amount after UV light irradiation.

744

Captions of tables

745

Table 1. Effect of phosphorylation degree on size and zeta potential of P-Zein.

746 747

Table 2. Effect of phosphorylation degree and pH on size, zeta potential, and encapsulation efficiency of AVM@P-Zein.

748

Table 3. Fitting results for the data of sustained release of AVM at different pH values.

749 750

Table 4. Results of toxicity analysis for AVM dispersing in ethanol solution and AVM@P-Zein.

38


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751


Graphic for table of contents hydrophobic drug

hydrophobic core

(AVM)

(Zein)

Zein pH=13

Phosphorylation water 752

hydrophilic shell (STP)

Zein disperse in water STP

39


water

Phosphorylated zein as biodegradable and aqueous nano-carriers for

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