Sulforaphane Loaded PEGylated Iron Oxide-Gold ...

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Sulforaphane Loaded PEGylated Iron Oxide-Gold Core Shell Nanoparticles: A Promising Delivery System for Cancer Therapy Hamidreza Kheiri Manjili,1,2Leila Ma’mani,3Amir Izadi,4 Elham Moslemi,5Maedeh Mashhadikhan,6Majid Mossahebi Mohammadi,7ShararehTavaddod,8 Abbas Shafiee,9Hossein Naderi-Manesh8* 1

Department of Nanotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran; Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; 3 Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran; 4 Young Researcher Club and Elite club, Islamic Azad University, East Tehran Branch, Tehran, Iran; 5 Department of Biology, School of Basic Sciences, Islamic Azad University, East Tehran Branch, Tehran, Iran;6Departments of Biology, Faculty of Science, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran;7Departments of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran; 8Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran;9Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran. *CorrespondingEmail address:[email protected]

2

Abstract: Herein we described a novel combination of sulforaphane (SF) with PEGylated gold coated Fe3O4 magnetic nanoparticles [(PEGylated Fe3O4@Au) NPs] to promote SF maintenance as an effective and promising anticancer drug. Physico-chemical properties of synthesized MNPs were assessed by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, vibrating sample magnetometery, dynamic light scattering, and Fourier transform infrared spectroscopy.Our in-vitro study of the effects of SF loaded (PEGylated Fe3O4@Au)on cell viability in a human breast adenocarcinoma cell line (MCF-7) confirms that SF cytotoxicity augmented when it was loaded onto the (PEGylated Fe3O4@Au) NPs compared with to SF alone or bare NPs. Further results of our flow cytometry and real-time PCR analyses established that this combination increased therapeutic effects of SF to induce apoptosis and necrosis, in addition to inhibiting migration in MCF-7 cell line. Our future experiments may introduce Fe3O4@Au NPs as a smart delivery system for SF for use in preclinical and in-vivo studies.

Keywords: Apoptosis, Breast cancer, Cancer therapy, PEGylated gold-coated Fe3O4 nanoparticles, Sulforaphane.

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region[20], where the biological tissues are transparent [21-

INTRODUCTION Over the past 30 years, many efforts have been made to

22]. Due to advantageous characteristics including its super

synthesizes and modify nanoparticles (NPs) for biomedical

paramagnetic property, non-toxicity[23], inertness, ease of

applications [1-3]. In recent decades, magnetic nanoparticles

detection in the human body[24], and high biocompatibility

(MNPs) have attracted a great deal of attention due to their

[25], gold-coated MNPs have been used for a variety of

potential biomedical uses[4-6]. MNPs, are 1-100 nm in

biological

diameter [7], and have been used for therapeutic targets such

introduction of novel low-cost and efficient anticancer DDSs

as cancer treatment [8, 9], diagnosis[10], magnetic resonance

integrating magnetic gold coated iron oxide NPs with various

imaging (MRI) contrast agents [11], magnetic cell separation,

surface characteristics would be of great significance [28,29].

and immunoassays. Directed drug delivery using MNPs is an

Consequently, focusing on valuable SF medicinal properties

attractive technique to improve the performance of cancer

and continuing our interest in the field of nanomaterials[30],

therapy [12]. Safe and non-toxic formulations of drugsthat

we present an efficient nano-drug delivery system to enhance

specifically destroy cancer cells without damaging normal

the absorption and therapeutic level of SF in the breast cancer

cells have become the goal of recent research. Drug delivery

cell line MCF-7. Presumably, the SF loaded (PEGylated

systems (DDSs) have been most extensively explored using

Fe3O4@Au) NPs may be a good candidate for the delivery of

various nano-devices which can be precisely fabricated with

SF for breast cancer treatment.

applications

[14a,

26,27].

Therefore,

the

various characteristics such as proper size, shape, surface charge, and stability for in vitro and in vivo applications.

MATERIALS AND METHODS

Among them, one of the most used MNPs is magnetic iron Cell lines, cell cultures, and characterization experiments

oxide (Fe3O4) due to its fine size and biocompatibility[13]. Sulforaphane (SF) is a chemopreventive agent, which can reduce, postpone, or reverse the process of carcinogenesis and is known as a promising and powerful anti-carcinogen in various types of cancers [14]. SF is an isothiocyanate compound

(chemically

named

1-isothiocyanato-4-

(methylsulfinyl)-butane) found in broccoli, cauliflower, kale and other cruciferous vegetables. Because of SF instability and sensitivity to oxygen, heat, and alkaline conditions, it is difficult to manufacture and distribute SF in food and pharmaceutical industries [15,16]. It has been reported that SF induces

its

cancer

chemoprotection

through

various

mechanisms, including inducing apoptosis and cell cycle arrest, inhibiting angiogenesis and cancer metastasis [17], antiinflammatory activity and inhibiting cytochorome P450 activity following activation of deoxidification enzymes [18]. In brief, SF potently prevents angiogenesis of tumors and metastasis

by

dropping

microcapillary

formation

and

inhibiting cell migration [19]. Gold NPs (Au NPs) have been well studied because of their large optical adsorption coefficients in the near-infrared

The human breast adenocarcinoma cell line MCF-7was purchased from the National Cell Bank of Iran (Pasteur Institute, Iran). The cells were cultured in Dulbecco's modiﬁed Eagle's medium (DMEM) (Gibco, Germany) supplemented with 2 mM L-glutamine, penicillin (50 IU/mL), streptomycin (50 µg/mL) and 10% Fetal Bovine Serum (FBS) (Gibco, Germany), incubated at 37º Cin a humidified incubator with5% CO2 atmosphere. Cells were dissociated using 0.53 mM EDTA in phosphate-buffered saline (PBS) and 0.25% trypsin. To inspect NP sizes, the samples were observed by transmission electron microscopy(TEM) (Hitachi H-7650, Tokyo, Japan) at the voltage of 80 kV and images of morphological characteristics obtained by scanning electron microscopy(SEM)

(Hitachi

S-4800

II).

X-ray

diffraction(XRD) data were measured by a Philips X’pert 1710 diffractometer using Cu Kα (α=1.54056 Å) in BraggBrentano geometry (θ-2θ) to determine the composition structure of NPs, and magnetic properties were determined with vibrating sample magnetometery(VSM). TheFourier transform infrared spectroscopy (FTIR) spectra were taken using a Nicolet FT-IR Magna 550 spectrophotometer (USA)



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in the region of 4000–400 cm . Aggregation levels of Fe3O4

concentrations was used to determine the SF loading

compared with Fe3O4@Au were investigated under an

efficiency.

inverted microscope (Olympus, IX81) and digital images were captured with a DP72 CCD camera.

The

release

of

SF

from

SF-loaded

(PEGylated

Fe3O4@Au) NPs was measured at 2-120 h and at different pH levels, including 7.4 and 5.4. PBS and citrate buffer were used for providing pH 7.4 and pH 5.4, respectively. Therefore, 20

Synthesis of PEGylated Fe3O4@Au NPs With some modifications, Fe3O4 and Fe3O4@Au NPs were synthesized according to the previous descriptions

31

mg of SF loaded (PEGylated Fe3O4@Au) NPs dissolved in 5

.

mL of PBS or citrate buffer and added into the dialysis bags

Briefly, a 2:1 ratio of ferric and ferrous chloride (2M) was

(molecular weight cut-off of 14 kDa) at 37°C and 100 rpm.

dissolved in deionized water, and the pH reached 10 by adding

After each time point, 2 mL of samples were collected and

ammonia on a stirrer. This was followed by 1 h stirring at

replaced with 2 mL of same fresh buffer solution. Drug

room temperature (RT) plus 30 min at 80˚C, until the dark

releases at different time intervals (during 5 days) were

MNPs appeared in the solution. An external magnetic field

measured using a UV-visible spectrophotometer at 235 nm.

was applied to separate Fe3O4 NPs from the solution. Then, a 2% solution of Fe3O4 NPs was prepared in sodium citrate (10 mM). This solution was sonicated and slowly heated to 70˚C on a stirrer. Then, the 0.1 M solution of HAuCl4.4H2O was dropped into the solution and stirred vigorously for approximately 30 min, allowing Au3+ to attach to the surface of the MNPs. Finally, it was cooled by stirring at RT, and a magnetic field was used again to purifye the Fe3O4@Au from Au NPs. Further, 0.04 g of Fe3O4@Au dispersed in 5 mL of CHCl3 was added to 0.03 g thiolated polyethylene glycol (HSPEG-OMe)in CHCl3. The mixture was stirred for 2 h under Ar gas, in the dark at RT. The particles were then collected by applying an external magnetic device or centrifugation and washed with CHCl3 and hexane (1:5 V/V). Then to obtain pure (PEGylated Fe3O4@Au) NPs, the resulting product was dialyzed for 24 h.

MTT assay MTT assays were performedto study the effects of SFloaded (PEGylated Fe3O4@Au) NPs versus free SF on cell proliferation and viability. MTT [3-(4,5-dimethylthiazol-2-yl) 2, 5-iphenyltetrazolium bromide] is a quantitative colorimetric test based on the ability of live cells to reduce the yellowcolored MTT by their mitochondrial enzyme succinate dehydrogenase to the MTT formazan crystals, which are dark blue. Therefore, the MCF-7 cells were cultured (1×105 cells/well) in 96-well plates with 200 μL DMEM. After 12 h, the fresh medium containing different doses (0.75-24 μmol/L in DMEM) of free SF was applied to cells and incubated at 37 °C with 5% CO2. In addition, it was repeated for equivalent concentrations of SF-loaded (PEGylated Fe3O4@Au) NPs and bare NPs plus the cultured cells without any treatment as a control. After 72 h, 20µL of MTT solution (5 mg/mL) was

SF loading and release efficiency

added, and the plates were incubated for 4 h at 37˚C. Then, the

For the purpose of measuring the SF loading efficiency,

formazan crystals were dissolved by adding DMSO (100

the optical density of an SF aqueous solution was measured at

μL/well) which left at 37 °C for 15 min. The optical density

235 nm using UV-visible spectrophotometry. Therefore, an SF

was measured at 570 nm using a micro-plate reader (Biotek,

solution with the primary concentration of 0.05 mg/mL was

USA). All the experiments were performed in triplicate.

mixed with 1 mg of (PEGylated Fe3O4@Au) NPs content by a pH 7 buffer of Tris/HCl. After 1 and 3h, a magnetic field was used externally to separate NPs (which may contain SF) from SF solutions. The difference between initial and residual SF

Extraction of total RNA and cDNA synthesis According to the manufacturer’s instructions (Invitrogen, USA) of TRIzol reagent the total RNA was extracted from MCF-7 cells 72 h after the treatment with different systems, including free SF, [PEGylated Fe3O4@Au], and SF loaded



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(PEGylated Fe3O4@Au), NPs. The A260/A280 ratio of

deviation (SD); values of P