Selenium nanoparticles as a carrier of 5-fluorouracil to achieve ...

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Selenium nanoparticles as a carrier of 5-fluorouracil to achieve anticancer synergism

Wen Liu †,#, Xiaoling Li †,#, Yum-Shing Wong ‡, Wenjie Zheng †,*, Yibo Zhang †, Wenqiang Cao †, Tianfeng Chen †,*



Department of Chemistry, Jinan University, Guangzhou 510632, China



School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China

*

Corresponding author: Tel: +86 20-85225962; Fax: +86 20 85221263.

E-mail: [email protected] (T. Chen); [email protected] (W. Zheng).

#

Authors contributed equally to the work.

Materials and methods In Vitro Drug Release 10 mg of 5FU-SeNPs were suspended in 10 ml of PBS (pH = 7.4) with constant shaking at 37˚C in a hard glass tube. At specific time following incubation, specific amount of PBS was taken out from the vial with pipette and the same volume of fresh PBS was replaced. All samples were analyzed using HPLC system (Agilent 1100) with a Model UV-1000 UV detector, and the detection wavelength was set at 266 nm. The column used was µ-BondapakTM C18 (4 × 300 mm, Grom, Germany). Mobile phase was acetate buffer solution (pH = 4.7) and flow rate was adjusted at 1.0 ml/min.

Acute toxicity at non lethal dose Eighty-eight mice were randomly divided into eleven groups with eight mice per group. The mice orally received saline (control), SeM or 5FU-SeNPs at the dose of 10 mg Se/kg, respectively. The mice in treated groups were sacrificed at 6, 12, 24, 36 and 48 h, and then subjected to the analysis of enzyme activities.

Determination of ALT and AST activities The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined by spectrophotometry using assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, P.R. China). The mice livers were homogenized with ice cold saline and centrifuged at 12,000 g at 4℃ for 30 min. The obtained supernatants were used for analysis of ALT and AST activities. The protein concentrations in the cell lysates were determined by bicinchoninic acid assay (Sigma) according to the manufacturer’s instructions.

Results and discussion

A

O 1s

B

N 1s 5FU-SeNPs

5FU-SeNPs

5FU

528 530 532 534 536 538 540

5FU

396

398

Binding energy (eV)

400

402

404

406

Binding energy (eV)

Figure S1. XPS spectra of O 1s (A) and N 1s (B) in 5FU and 5FU-SeNPs. In the fitting spectrum of O 1s (A), the peak in 5FU-SeNPs split into two ones. The one at 532.2 eV possibly comes from the molecule of 5FU, and its shift to 531.4 eV may correspond to the formation of Se-O band. The change (∆eV=0.21 eV) in N 1s peak between 5FU and 5FU-SeNPs in (B) supports the formation of Se-N band.

Fluorescence Intensity

600

5FU-SeNPs SeNPs

500 400 300 200 100 0

500

550 600 650 Wavelength (nm)

700

700

C 700

600

600

Fluorescence Intensity

B

700

Fluorescence Intensity

A

500 400 300

5FU-SeNPs SeNPs

200 100 0

500

5FU-SeNPs SeNPs

400 300 200 100

520

560 600 640 Wavelength (nm)

680

0

500

550 600 650 Wavelength (nm)

700

Figure S2. Fluorescence spectra of 6-coumarin loaded 5FU-SeNPs and SeNPs in DMSO (A) and in PBS, pH 7.4 (B) and 0.2 M NaOH solution containing 0.5% Triton X-100 (C) with the excitation wavelength set at 485 nm. Nanoparticles were collected by centrifugation at 8000×g for 20 min in a clean microcentrifuge tube and re-suspended in different solutions. Then the spectra were recorded on a Cary Eclipse spectrofluorometer (Varian; Palo Alto, CA). 6-coumarin loaded 5FU-SeNPs and SeNPs have the similar fluorescence intensity in PBS, DMSO and 0.2 M NaOH solution containing 0.5% Triton X-100. This results directly confirm that 5FU-SeNPs and SeNPs have the same fluorescent labeling efficiencies, thus could be use in fluorescent readout as an indicator of nanoparticle cellular uptake.

Cellular uptake (μg/106 cells)

40

*

30

**

20 10 0

5FU-SeNPs +

+

+

+

5FU (μg/ml) -

1

5

10

Figure S3. Quantitative analysis of the effects of 5FU on the cellular uptake efficiency of 6-coumarin loaded 5FU-SeNPs (40 µg/ml) in A375 cells after 2-h incubation. Values expressed are means ± SD of triplicates. Significant difference between treatment and control groups is indicated at P