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Engineering Multifunctional Coatings on Nanoparticles Based on Oxidative Coupling Assembly of Polyphenols for Stimuli-Responsive Drug Delivery Hongshan Liang, Bin Zhou, Jing Li, Xingnian Liu, Ziyu Deng, and Bin Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01208 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Journal of Agricultural and Food Chemistry

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Engineering Multifunctional Coatings on Nanoparticles Based on Oxidative

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Coupling Assembly of Polyphenols   for Stimuli-Responsive Drug Delivery

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Hongshan Liang a,c, Bin Zhou b, Jing Li a,c, Xingnian Liu a,c, Ziyu Deng a,c, Bin Li a,c,d*

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a

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430070, China

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b

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Wuhan 430068, China

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c

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University), Ministry of Education, China

College of Food Science and Technology, Huazhong Agricultural University, Wuhan

School of Food and Biological Engineering, Hubei University of Technology,

Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural

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d

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China

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*Corresponding author: Bin Li

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E-mail address: [email protected]

Functional Food Enginnering & Technology Research Center of Hubei Province,

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ACS Paragon Plus Environment


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Abstract

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In this study, zein nanoparticles (NPs) with novel multifunctional coatings based on

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oxidative coupling assembly of polyphenols were synthesized for the first time. This

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coating was formed by oxidative self-polymerization of the organic ligands

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(polyphenols) in alkaline condition, which could be biodegraded by acidic pH, as a

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result, impacting pH-responsive property of the system. More importantly, the high

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level of intracellular glutathione (GSH) could induce the biodegradation of the

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polyphenol coatings, resulting in a fast release of trapped anticancer drugs in the cells.

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Based on confocal laser scanning microscopy (CLSM) and cytotoxicity experiments,

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drug-loaded and polyphenol-coated zein NPs were shown to possess highly efficient

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internalization and an apparent cytotoxic effect on HeLa cells. Notably, the CLSM

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observation illustrated that coated zein NPs showed delayed drug release compared

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with free drug or DOX-loaded zein NPs without coatings, resulting from the

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pH-responsive release of loaded drug in extra/intra-cellular environment. Additionally,

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the short-time cytotoxicity and morphology observation also confirmed the delayed

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drug release behavior of coated NPs. These highly biocompatible and biodegradable

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polyphenol-coated zein NPs may be promising vectors in the field of

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controlled-release biomedical applications and cancer therapy.

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Key words: polyphenol, self-polymerization, pH-responsive, redox dual-responsive

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1. Introduction

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Encapsulation of guest molecules within micro/nano-sized hosts provides a variety of

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promising applications in the fields of catalysis 1, medical diagnostics 2, drug delivery

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3, 4

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emulsions,

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nanoparticles (NPs) have attracted considerable attention as potential drug delivery

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devices

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light- 11 and temperature- 12 responsive, is now a key theme in the biomedical field for

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effective loading and release of guest molecules with high specificity to targeted cells

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in a controlled manner.

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Protein-based systems represent a major class for drug and gene delivery due to their

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enhanced properties of absorbability and low toxicity in the degradation of end

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products

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candidates for drugs or bioactives delivery

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controlled release systems have been developed by employing different kinds of

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coatings or stabilizers to improve the stability or loading capacity of bulk zein NPs

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19-21

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based on zein NPs or zein/quaternized chitosan NPs capped with metal

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ion-polyphenol coatings

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used as a stimuli-responsive mechanism to trigger drug release. Despite these

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designable pH-responsive delivery systems, current zein-based drug delivery systems

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still have a number of flaws, including for example low stimulus sensitivity especially

and materials science 5. Among the various hosts/carriers including micelles, hydrogel

and

inorganic

particles,

self-assembled

6-8

biodegradable

. Engineering stimuli-responsive functional NPs, e.g., pH- 9, redox-

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,

13-15

. Of protein-based NPs, zein NPs have been highlighted as excellent 16-18

. Additionally, several zein-based

. In our previous work, we reported a bio-responsive controlled-release system

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. The metal ion-polyphenol coordination bonding was

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in mild acidic, the relative slow release of the drug when entering the cell and

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introducing potential toxic species. Therefore, the development of a green,

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biocompatible, and effective drug delivery system with high stimulus sensitivity and

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intracellular controlled-release features remains a desirable goal.

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Plant polyphenols are widely distributed in plant tissues and plays a critical role in a

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range of biological functions such as photosynthesis, structural support, oxygen

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transportation and adhesion 24, 25. With the high content of dihydroxyphenyl (catechol)

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and trihydroxyphenyl (gallic acid), plant polyphenols display versatile physical and

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chemical properties, including absorption of UV radiation, radical scavenging, and

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metal ion complexation

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based on these properties of polyphenols in the field of chemistry and materials

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science. Very recently, plant polyphenols have been investigated as precursors for the

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formation of multifunctional coatings on different substrates 25, 26. Coating deposition

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was most effective from alkaline condition (0.6M NaCl, pH 7.8) as compared to pure

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water, resulting from the oxidation of plant polyphenols leading to the self-polymerize

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27, 28

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polyphenols 28.

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Due to the unique properties of polyphenols, we engineered multifunctional coatings

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on zein NPs based on oxidative coupling assembly of polyphenols. Tannic acid (TA),

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which contains high amount of galloyl groups, was first used as the model polyphenol

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precursor for the deposition of the coatings. The degradation of the coatings was

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tailored by the pH conditions and intracellular glutathione (GSH) level, leading to

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. Significant interest has been given to building blocks

. Notably, thiolysis reaction could lead to the degradation of polymeric

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drug release and, hence, cellular apoptosis. This simple coating method without any

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other organic reagents or cross-linkers met the requirements for a variety of

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biomedical applications.

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2. Materials and methods

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2.1. Materials

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Zein (Z0001) was purchased from Tokyo Chemistry Industry, Co., Ltd. (Tokyo,

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Japan). Tannin acid (TA) was purchased from Aladdin Chemistry Co., Ltd.

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(-)-epigallocatechin-3-gallate (EGCG) was purchased from Xi'an Natural Field

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Bio-Technique Co., Ltd. (Xi’an China). Persimmon tannin (PT, 98.7% purity) was

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kindly provided by Shanghai Ocean University composed of polymers ranging from 7

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to 20 kDa. 3-(N-morpholino)-propanesulfonic acid (MOPS) was obtained from the

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Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Glutathione (GSH),

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phosphate buffer solution (PBS) and MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl

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tetrazolium bromide] were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Dubelcco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),

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trypsin-EDTA and penicillin-streptomycin mixtures were from Gibco®BRL (Carlsbad,

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CA, USA). Other chemicals used were of analytical grade. All the solutions used in

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the experiments were prepared using ultrapure water through a Millipore (Millipore,

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Milford, MA, USA) Milli-Q water purification system.

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2.2. Preparation and characterization of polyphenol-coated zein NPs

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Preparation of polyphenol-coated zein NPs: All solutions were freshly prepared for

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immediate use. The standard preparation process was described as follows: zein was

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dissolved in aqueous ethanol solutions (75% v/v) to obtain a stock solution with final

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concentration of 10 mg/mL. Then 0.5 mL zein solution was added to 9.5 mL of

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MOPS buffer (10 mM, pH 7.8). Next, different volume (40 µL, 60 µL, 80 µL or 100

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µL) of TA solution (24 mM) was added and the dispersion was under vigorous stirring

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for 0.5-2 h at room temperature. The product was then purified by successive dialysis

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(MWCO 3500) against deionized water for 48 h to remove the free TA. Then 200 µL

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of HAuCl4 aqueous solutions (10 mM) was added in the zein-TA solution to observe

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the metallization of gold.

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Dynamic laser scattering (DLS) and zeta potential: DLS and zeta potential

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measurement were performed on a commercial laser light scattering instrument

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(Nano-ZS90, Malvern, UK). The apparent Z-average hydrodynamic diameter and

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polydispersity index (PDI) were obtained at 25°C with a fixed scattering angle of

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173°. The zeta potential was calculated by the Dispersion Technology Software.

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Transmission electron microscope (TEM): Images were taken on a JEM-2100F

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(JEOL, Japan). The samples were prepared by dropping solution onto copper grids

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coated with carbon and then dried naturally.

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Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron

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spectroscopy (XPS) analysis: FT-IR spectra were obtained with a Jasco 4100 series

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with an attenuated total reflection cell (Jasco Inc., Easton, MO). All samples were

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prepared as KBr pellets and were scanned against a blank KBr pellet background.

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XPS observations were conducted on an axis ultra DLD apparatus (Kratos, U.K.).

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2.3. DOX encapsulation

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The stock of 4 mg/mL DOX prepared was completely dissolved in zein solution for

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60 min. The formulation containing DOX was prepared by adding the above solution

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dropwise to MOPS buffer (10 mM, pH 7.8) with magnetic stirring. Next different

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volume (40 µL, 60 µL, 80 µL or 100 µL) of TA solution (24 mM) was added and the

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dispersion was under vigorous stirring for 0.5-2 h at room temperature. The free DOX

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was obtained by calculating the DOX content that were ultracentrifuged at 4000 × g

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for 30 min in a refrigerated centrifuge (TGL-20000cR) with angle rotor through 10

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kDa MWCO Amicon filter and determined by a UV-vis spectrophotometer (UV-1100,

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MAPADA) at 480 nm. The encapsulation efficiency was defined as the drug content

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that was entrapped into zein/TA NPs and calculated as follows:

EE (%) =

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Total DOX - Free DOX × 100 % Total DOX

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2.4. Release of DOX

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Drug release from NPs and release kinetics study were carried out using dialysis

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membrane tubing (MWCO = 3500). Briefly, an aliquot of drug loaded NPs was taken

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into a dialysis bag and suspended in 100 mL release medium with different pH values

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(pH = 4.0, 5.0, 6.2, 7.4) or different GSH concentrations at room temperature and

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gently shaken at 100 rpm in a water bath (37.0 ± 0.5 °C). At predetermined intervals,

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1 mL dissolution sample was collected and the concentration of DOX was measured.

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After that, each 1mL aliquot was returned to the original solution for maintaining total

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volume and total DOX amount.

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2.5. In vitro cell toxicity assay

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The cytotoxicity of free DOX and coated NPs was evaluated using the MTT assay. 7



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Briefly, human cervical cancer cells (HeLa cells) were seeded in 96-well microplates

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at the density of 1 × 104 cells/well and incubated at 37 °C under a 5% CO2 atmosphere

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for 24 h to allow cell attachment. After incubation, the medium was replaced by the

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fresh medium containing various NPs (either blank or DOX containing NPs) at DOX

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concentrations ranging from 0.1 to 2.5 µg/mL for further incubation of 24 h. Then 20

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µL MTT solution (5 mg/mL) was added to each well, and the cells were incubated for

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another 4 h. After incubation, the culture solution was removed carefully, leaving the

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precipitate. Subsequently, 100 µL of DMSO was added to each well to solubilize the

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formazan crystals formed. The absorbance at 490 nm was measured by a multilabel

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microplate reader (Victor X3, PerkinElmer 2030). Cell viability (%) was expressed by

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the following equation:

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Cell viability (%) =

Abs 490 nm of treated group × 100 % Abs 490 nm of control group

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2.6. Intracellular uptake study

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For quantitative study, HeLa cells were seeded in a 6-well plate at a density of 2×105

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cell/well in 2 mL growth medium and the cells were incubated at 37 °C for 24 h to

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allow cell attachment. Then the culture medium was replaced by 2 mL of fresh

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medium containing DOX-loaded NPs and incubated for 1, 2, 4 or 6 h, respectively.

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After incubation, the suspension was collected and the wells were washed three times

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with cold PBS and harvested. The cellular uptake of NPs was measured by flow

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cytometry analysis (Beckman Coulter, Miami, FL, USA).

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For qualitative study, cells were seeded in a 35 mm petri dish at a density of 1×105

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viable cells/well in 2 mL growth medium and the cells were incubated at 37 °C for 24 8


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h to allow cell attachment. Cells were washed for three times after incubation for 1, 2,

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4 or 6 h using DOX-loaded NPs and then fixed by 4% paraformaldehyde in PBS (pH

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7.4) for 20 min. The cells were further washed twice with PBS. Fluorescence images

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were collected using a CLSM (Zeiss LSM 710, Germany).

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2.7. Morphology Observation

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The morphology of the cells after incubation of 24 h with blank or DOX containing

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NPs was evaluated by using optical microscopy with a 10×objective.

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2.8. Statistics analysis

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All of the data were expressed as the mean ± standard error. ANOVA analysis and

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Student’s t-test were performed to compare the significant difference. A value of p

Engineering Multifunctional Coatings on Nanoparticles Based on

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