Delivery of sesamol using polyethylene glycol-functionalized selenium

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Food and Beverage Chemistry/Biochemistry

Delivery of sesamol using polyethylene glycol-functionalized selenium nanoparticles in human liver cells in culture Fuguo Liu, Hua Liu, Runhua Liu, Chunxia Xiao, Xiang Duan, David Julian McClements, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06924 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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Journal of Agricultural and Food Chemistry

Cover letter Dear Editor, Please find the revised version of a manuscript entitled “Delivery of sesamol using polyethylene glycol-functionalized selenium nanoparticles in human liver cells in culture” that we submitted to Journal of Agricultural and Food Chemistry as a regular article. In this manuscript, we successfully prepared and characterized sesamol-loaded PEG selenium nanoparticles. We then showed that these nanoparticles can be used to effectively inhibit the growth of model cancer cells (HepG2).

These

nanoparticles may be useful for application in functional foods or medical foods designed to prevent or treat cancer. Thank you in advance for consideration of our manuscript. If you need any further information, please do not hesitate to contact us. Sincerely yours, Xuebo Liu College of Food Science and Engineering, Northwest A&F University, Yangling, China E-mail: [email protected]

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Delivery of sesamol using polyethylene glycol-functionalized

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selenium nanoparticles in human liver cells in culture

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Fuguo Liu†1, Hua Liu†1, Ruihua Liu†, Chunxia Xiao†, Xiang Duan†, David Julian

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McClements§, Xuebo Liu†*

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†College

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Shaanxi 712100, China

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§Department

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USA

of Food Science and Engineering, Northwest A&F University, Yangling,

of Food Science, University of Massachusetts, Amherst, MA, 01003,

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Journal: Journal of Agricultural and Food Chemistry

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Submitted: December 2018

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1Author

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*Corresponding author: Xuebo Liu, College of Food Science and Engineering,

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Northwest A&F University, 28. Xi-nong Road, Yangling 712100, China. E-mail:

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[email protected]; Fax: +86-029-87092325; Tel: +86-029-87092325

Contributions: F.L. and H.L. contributed equally to this work.

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ABSTRACT: Anticancer nanoparticles were fabricated by linking the nanoparticles

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of two known anticancer agents, sesamol and selenium, using polyethylene glycol

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(PEG). The successful fabrication of the sesamol-PEG-selenium nanoparticles

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(PEG-SeNPs), which had a sesamol loading efficiency of 10.0 ± 0.5 wt%, was

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demonstrated using different spectroscopic techniques. The impact of the

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nanoparticles on model cancer cells (HepG2) was established using cell activity test,

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morphological observation and fluorescent staining, which all showed the

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nanoparticles effectively inhibited the HepG2 cells. MTT assays showed the IC50 of

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PEG-SeNPs and sesamol-PEG-SeNPs on HepG2 cells was 413.8 and 68.7 μg/mL

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respectively, which indicated the synergistic inhibition between sesamol and selenium

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nanoparticles. Furthermore, flow cytometry showed the sesamol-PEG-SeNPs

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exhibited higher apoptosis than either sesamol or PEG-SeNPs alone. Finally, Western

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blot confirmed the apoptostic ability of sesamol-PEG-SeNPs was associated with

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downregulation of bcl-2, procasepase 3 and PARP, upregulation of Bax, and

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discharge of cytochrome c into the cytosol. Our findings suggest the novel

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sesamol-nanoparticles may be efficient anticancer agents.

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KEYWORDS: Polyethylene glycol; Selenium nanoparticles; Sesamol; Apoptosis;

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Anticancer

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INTRODUCTION Nanoparticles are increasingly investigated for their potential to increase the

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bioactivity and reduce the unwanted therapeutic side effects.1,

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nano-technology have been combined to establish a new generation of cancer

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treatments,

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drugs.5 As a result, researchers are examining a broad variety of nanoparticles for

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their potential advantages and disadvantages as therapeutics. 1, 6

3, 4

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Recently, bio- and

bringing about a critical therapeutic group of nanoparticle-based

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Recently, the incorporation of selenium (Se) into nanoparticles has attracted

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much attention because of its ability to promote tumor cell apoptosis. Se can sensitize

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tumor cells to antitumor drugs.7 Se nanoparticles (SeNPs) are more bioavailable and

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biocompatible and less toxic than conventional inorganic or organic Se compounds.8

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SeNPs can promote the apoptosis of cancer cells and therefore help prevent or reduce

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the incidence of cancers.9, 10 Moreover, SeNPs exhibit effective antitumor activity by

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hindering the growth or stimulating the death of various human cancer cells, including

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cells of hepatocyte (HepG2),11 breast cancer (MCF-7MDA-MB-231),12, 13 melanoma

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(A375)14 and cervical carcinoma (HeLa Hep-2).15 The selectivity towards tumor cells,

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small sizes and high permeability of SeNPs have brought about new methods for

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disease treatment. Moreover, SeNPs can be modified by many polymers to act as a

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gene and drug delivery system with different characteristics.16

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Polyethylene glycol (PEG) is an amphiphilic molecule that is well miscible with

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both water and various organic solvents.17,18 It also can overcome many 5



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physicochemical and chemical instabilities that sometimes limit the development of

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therapeutic applications, such as aggregation, adsorption, deamination, clipping, and

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oxidation.19 Therefore, PEG has become one of the most popular polymers for drug

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delivery.20 As reported, PEG-SeNPs induce mitochondria dysfunction to conquer drug

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resistance of hepatocellular cancer.11 Also, the lung cancer toxicity and apoptosis

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induction of PEG-SeNPs are enhanced after crocin conjugation.21

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Sesamol (3,4-methylenedioxy phenol), which results from sesamolin degradation,

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is physiologically resistant against oxidation, inflammation and cancers.22,23,24

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However, its usefulness may be limited by the low stability, oral bioavailability and

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fast elimination. There is therefore interest in designing sesamol nanoparticles to

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enhance its functionality.

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We designed a combined system of sesamol and SeNPs, which both have

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anticancer activities. Sesamol was surface-conjugated to SeNPs with the aid of PEG.

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Our study highlights the synergistic inhibition of model liver tumor cells and throws

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light on the possible mechanism for apoptotic pathway by measuring cellular reactive

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oxygen species (ROS) and mitochondrial membrane potential (MMP) and through

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Western blot.

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MATERIALS AND METHODS

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Chemicals. Na2SeO3 (Tianjin Fuchen Chemical Reagents Factory); polyethylene

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glycol (PEG)200 (Tianjin KeMiOu Chemical Reagent Co. Ltd); sesamol (98%,

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S3003), acridine orange (AO), ethidium bromide (EB), and 40,6-diamidino6


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2-phenylindole (DAPI, Sigma-Aldrich Chemicals Company, USA); annexin V-FITC

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apoptosis detection kit, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine

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iodide (JC-1), ROS assay kit and thiazolyl blue tetrazolium bromide (MTT)

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(Beyotime Biotechnology Company) were all of regent grade and used as received.

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Synthesis of PEG-SeNPs. PEG-SeNPs were prepared as reported with a bit

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modification.11 Briefly, 5 mL of Milli-Q water containing 4.35 mg of Na2SeO3

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powder was added with 10 mL of a PEG200 solution and heated at 210-220 °C in a

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well-stirred silicone oil bath for about 18 min under continuous magnetic stirring. The

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resulting substances were blended with water (1:1), centrifuged at 12,000 rpm for 5

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min and then washed with Milli-Q water three times to get rid of excessive PEG. The

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products were observed spectroscopically and microscopically.

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Preparation of sesamol-conjugated PEG-SeNPs. According to a reported

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approach with modification,25 a 5-mL aliquot of PEG-SeNPs was added with 5 mL of

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32.5 mg/mL sesamol and finally diluted to 12.5 mL with Milli-Q water under stirring

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at room temperature for 48 h. Excessive sesamol was deleted by dialysis in a mixture

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of PEG and Milli-Q water at a volume ratio of 1:5. Then the products were

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characterized as described later.

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Characterization. The spectroscopic techniques included an ND-2000

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UV-visible spectrophotometer (Thermo Fisher Scientific, Inc., USA), a VERTEX 70

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Fourier transform infrared spectroscope (FTIR, Bruker Optics, Ettlingen, Germany),

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and a PHI Quantera X-ray photoelectron spectrometer (XPS, ULVAC-PHI, INC,

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Japan). Surface morphology of the nanoparticles was tested under a field-emission

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scanning electron microscope (Hitachi S-4800, Japan). The loading percentage of

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sesamol within the PEG-SeNPs was estimated based on the sesamol content in the

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centrifuged pellets, which was determined by measuring the UV absorbance at 298

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nm using UV/visible spectroscopy as follows: Loading efficiency =w0/w×100%

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where w0 and w are the weights of sesamol initially and conjugated to the

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PEG-SeNPs, respectively.

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Cell cultures. Human hepatoblastoma HepG2 cells and human hepatocyte cells

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HL7702 from the 4th Military Medical University (Xi’an, China) were incubated in

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RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in

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a humid incubator (5% CO2).

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Detection of cell viability and cytotoxicity. Cells (1 × 106 cells/mL) were

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cultured in 96-well polystyrene plates at 37 °C and 5% (v/v) CO2 for 24 h. Then 100

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L of the medium each well was added with 100 L of sesamol, PEG-SeNPs or

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sesamol-PEG-SeNPs at specific contents and incubated for 12 or 24 h separately.

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After that, each well was added with 100 μL of 0.5% (w/v) MTT for 4 h of culture,

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followed by absorbance detection at 490 nm on a microplate reader (Bio-Rad, China).

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Cell viability was estimated as a percent relative to the control cells.

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AO-EB and DAPI staining assays. AO emits a green fluorescence and embeds

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in nuclear DNA if the cell membrane is complete, while the red–orange light of EB

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indicates nuclear DNA damaged cells.26 Thus, the apoptotic cells were qualitatively

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observed

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sesamol-PEG-SeNPs were added into 60 μL of AO/EB (100 μg/mL for both dyes)

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and immediately imaged under an IX71 inverted fluorescence microscope with a

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DP70 digital camera (both Olympus, Tokyo, Japan). For DAPI staining,

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sesamol-SeNPs- treated HepG2 cells were processed first with 4% formaldehyde for

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10 min and then with 1 μg/mL DAPI for 5 min before microscopic observation.

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after

Detection

AO/EB

of

staining.

cellular

In

brief,

ROS.

After

the

HepG2

cells

incubation,

10

treated

μM

by

of

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2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma) was added to the

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wells at 37 °C for 30 min and the resulting color was quickly detected by the

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fluorescence microscope and a microplate reader (Molecular Devices, USA).

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Measurement of MMP. JC-1 can selectively enter mitochondria to form

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monomers. If MMP collapses during apoptosis, the fluorescence of JC-1 turns from

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red to green, and thus the green and red ratio of JC-1 indicates the variation of MMP.

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The test cells were added with 100 L of 5 μg/mL JC-1 for 30 min and then

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quantified by the fluorescence microscope and microplate reader.

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Flow cytometry. Cells were collected, resuspended to 106 cells/mL in the binding

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buffer (10 mM Hepes / NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2), and then

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cultured with FITC-annexin V and propidium iodide (PI) at room temperature for 15

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min, followed by apoptosis assay by FACSDiVa (BD Biosciences).

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Western blot. The harvested cells were lysed with a P0013 cell lysis buffer

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(Beyotime), followed by total protein detection by a BCA kit (ThermoFisher). After

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were

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electronically removed onto polyvinylidene fluoride membranes (0.45 um, Millipore)

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through a semi-dry instrument (Bio-Rad). After 2 h of blocking in 5% non-fat dry

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milk in TBST (20 mM Tris, 166 mM NaCl, and 0.05% Tween 20, pH 7.5), the

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samples were cultured first with primary antibodies overnight at 4 °C and then with

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secondary antibodies at 25 °C for 2 h. Blots were formed in a luminescent substrate

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(ThermoFisher) and exposed on a molecular imager ChemiDoc XRS device

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(Bio-Rad).

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Statistical analysis. Each measurement was repeated three times. Results were

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assessed via analysis of variance and post-hoc Fisher's least significant difference test

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at the significant level p ≤0.05. Data were shown as mean ± standard deviation.

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RESULTS AND DISCUSSION

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Synthesis and characterization of nanoparticles

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UV-vis spectroscopy. The formation of PEG-SeNPs was validated via UV-vis

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spectroscopy (Fig. 1A). The PEG-SeNPs exhibited a maximum absorption at 210 nm,

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suggesting SeNPs typically have a maximum absorption at 200 to 400 nm.12 A similar

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maximum absorption was reported in SeNPs prepared from lemon leaf extract and

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Gliocladium roseum.27, 28 In addition, since sesamol has a peak at about 298 nm in the

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UV-visible region29, its conjugation onto the NPs also displays the typical peaks of

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sesamol at 293 and 231 nm. 10


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FTIR analysis and valence state of selenium. The variations in chemical

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bonding through the generation of sesamol, PEG-SeNPs or sesamol-PEG-SeNPs were

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characterized via FTIR analysis (Figure 1B). Peaks characteristic of the PEG

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functional groups at 2874.2 and 1103.9 cm-1 were ascribed to -CH

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respectively. PEG-SeNPs display the bands at 3366, 2922 and 1072 cm-1 assigned to

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the stretching (n) vibration of -O-H, -C-H and -C-O-C, respectively, suggesting the

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attachment of PEG to the SeNPs. In the spectrum of the sesamol-PEG-SeNPs, the

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bands at 3435 and 2924 cm-1 shifted slightly compared with PEG-SeNPs found at

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3366 and 2922 cm-1. Moreover, the band at 1094 cm-1 was widened from the

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superposition of bands at 1188, 1043 cm-1 (from sesamol) and 1072 cm-1 (form

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PEG-SeNPs). Also the bands at 1462, 1394 and 1246 cm-1 can be associated to the

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conjugation of sesamol onto the PEG-SeNPs, which shifted slightly compared with

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sesamol (1479, 1391 and 1264 cm-1 respectively). Thus the FTIR spectra are also

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consistent with the formation of sesamol-PEG-SeNPs. Since elemental selenium is

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less toxic and more bioactive than its other forms32 (Fig. 1C), the Se 3d and 3p peaks

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at 56 and 180 eV respectively indicate Se was in the valence state of zero (Se0) and

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the C1s and O1s peaks at 285 and 532 eV respectively indicate PEG was successfully

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connected to Se.

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and -C-O-C,31

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Loading profile and in vitro release of sesamol. The feasibility of PEG-SeNPs

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as bioactive carriers was assessed by calculating the loading efficiency of sesamol

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onto the nanoparticles, which was found to be 10±0.5 wt%. Since the absorption of

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nanoparticles is accomplished by cell endocytosis and the first site to be reached is the

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lysosome,33 the release rate test of sesamol-PEG-SeNPs was simulated at the pH of

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cell lysosome (pH=5.4). The release rate of sesamol reached 65.4.4% at 24 h and

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84.7% at 48 h (Fig. 1D), indicating the nanoparticles reduced the release rate of

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sesamol and played a role in sustaining the release.

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Appearance and morphological analysis of sesamol nanoparticles. The

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appearance in Fig. 1E shows the color change during the nanoparticle formation. The

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colloidal solution which was initially colorless turned red after reduction with PEG,

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which was due to the surface plasma resonance. Similar color change was observed in

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SeNRs after chitosan stabilization34 or polyamidoamine modification.29 The color

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change observed in our study therefore confirms the successful production of SeNPs

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with PEG. In addition, the morphology and sizes of PEG-SeNPs and

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sesamol-PEG-SeNPs were characterized using an S-4800 PE-scanning electron

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microscope (Hitachi Japan; Fig. 1F&G). The nanoparticles were scattered and

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spherically-shaped. The diameter ranged from about 50 to 90 nm in PEG-SeNPs (Fig.

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1F) and slightly increased to about 100 nm in sesamol-PEG-SeNPs (Fig. 1G), which

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can be attributed to the conjugation of sesamol onto the NP surfaces. As reported, NPs

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in diameter above 200 nm are just deleted non-specifically by monocytes and the

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reticuloendothelial system35, while smaller particles (

Delivery of sesamol using polyethylene glycol-functionalized selenium

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