Electronic Supplementary information entitled

Facile fabrication of a novel 3D rose like lanthanum doped

zirconia decorated reduced graphene oxide nanosheets: An efficient

electro-catalyst for electrochemical reduction of futuristic anti-

cancer drug salinomycin during pharmacokinetic study

Saad A. Alkahtani1, Ashraf M. Mahmoud2,3, Mater H. Mahnashi3, Ramadan Ali4,

Mohamed M. El-Wekil*2

1Department of Clinical Pharmacy, College of Pharmacy, Najran University, Najran, Kingdom of Saudia Arabia.2Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt.3Department of Pharmaceutical Chemistry, College of Pharmacy, Najran University, Najran, Kingdom of Saudia Arabia.4Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assuit, Egypt.

* Corresponding author: mohamed.mohamoud@ymail.com

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1. Experimental

2.1. Materials and reagents

Salinomycin (SAL) monosodium salt was obtained as a gift from NODCAR, El-Dokky, El-Giza,

Egypt. Lanthanum nitrate (La (NO3)3.6H2O), Zirconyl chloride (ZrOCl2), dopamine, glutathione,

adenine, guanine and uric acid were purchased from Sigma Aldrich. Ascorbic acid, Na2HPO4 and

NaH2PO4 were purchased from El-Nasser for chemicals, Cairo, Egypt.

Phosphate buffers (0.2 M) with different pH values were prepared by mixing different volumes

of 0.2 M Na2HPO4 and 0.2 M NaH2PO4 and then, adjusting the pH with NaOH or H3PO4.

2.2. Instrumentation

Princeton VersaSTAT MC (VersaSTAT 3, Model RE-1, Princeton Applied Research, AMETEK,

USA), connected to three electrodes system was used for carrying out all investigated

electrochemical experiments such as cyclic voltammetry (CV), differential pulse voltammetry

(DPV) measurements. The reference electrode was Ag/AgCl, 3 M KCl and the auxiliary

electrode was a platinum wire, whereas the working electrode was the fabricated GCE in 10-mL

glass. Surface morphology studies of the manufactured sensor were investigated using scanning

electron microscopy (SEM, JEOL JSM-5400 LV instrument (Oxford, USA). All experiments

were conducted at room temperature. Elemental analysis was carried out using OXFORD INA

Energy Dispersive X-ray Spectrometer (EDX). Nicolet 6700 FTIR Advanced Gold

Spectrometer, supported with OMNIC 8 software (Thermo Electron Scientific instruments Corp.,

Madison, WI USA) was used to record FTIR spectra. Powder X-ray diffraction (PXRD) was

performed using a PW1729 Philips diffractometer interfaced with a computer control unit model

PW1710 using copper source. The angular range of 2θ was between 4º and 70º. Raman spectral

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studies were performed using Micro-Raman spectrometer (U.K) encompassed with 514.4 nm

He/Ne laser.

2.3. Synthesis of reduced graphene oxide (RGO)

Graphite oxide was synthesized from natural graphite by modified Hummers method. Briefly,

2.0 g of graphite powder and 1.0 g NaNO3 were mixed, and then put into 96.0 mL Conc. H2SO4

in an ice bath. Under vigorous stirring, 9.0 g KMnO4 was added gradually. The temperature of

the mixture was maintained below 20°C. The ice bath was removed and mixture was stirred in a

water bath for 2 h. To the brownish color pasty liquid, 150 mL of water was added. To maintain

temperature below 50°C water was added continuously till total volume of water was 200 mL.

To the above mixture, 5 mL of 30% H2O2 was added and it was observed that the solution color

transformed into brilliant yellow along with bubbling. The mixture was stirred for 2 h; it was

filtered and washed with 10% HCl aqueous solution, water, and ethanol. The product obtained

was dried under vacuum at 60°C. For the synthesis of reduced graphene oxide, 100 mg of

graphite oxide was dispersed in 100 mL of water and sonicated for 1h. In this step conversion of

graphite oxide to graphene oxide (brown dispersion) took place. To the above dispersion 2.0 g of

hydrazine hydrate in 5 mL water was added and the mixture was refluxed at 100 °C for 24 h

under magnetic stirring. Finally, the mixture was filtered, washed thoroughly with water and

dried at 60 °C for 12 h.

2.4. Preparation of real samples

2.4.1. Human urine and plasma samples

Urine samples were collected from healthy volunteers and 5 mL of urine sample was centrifuged

at 1500 rpm for 20 min. Then, the urine sample was filtered using 0.45 mm filter paper and 1.5

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mL of the supernatant was transferred to voltammetric sample containing phosphate buffer

(pH=7.0).

Human plasma (1.5 mL) was mixed with 1.0 mL acetonitrile and subjected to centrifugation to

about 30 min to remove possible interference. After that, the supernatant was collected and

diluted with 5 mL phosphate buffer (pH= 7.0) prior to the voltammetric analysis.

2.4.2. Pharmacokinetic studies in rabbit plasma

The purpose of this study was to investigate the pharmacokinetics of SAL. All experimental

procedures and protocols were reviewed and approved by the ethics of Assiut University clinics,

Assiut, Egypt. Rabbits had fed prohibited for 6 h before the study but water was only taken. The

blood samples (3.0 mL) were collected from a forearm vein into heparinized polyethene tubes at

0 (pre-dose), 0.5, 1.0, 1.5, 2, 2.2, 2.5, 3, 3.5, 4 and 5 h after oral administration of 2.5 mg mL -1,

the samples were immediately centrifuged at 4000 rpm for 10 min. The plasma was stored at

−80ºC until analysis. The pharmacokinetic parameters for SAL were estimated using the

validated moment analysis software.

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Scheme 1S. Conversion of GO into RGO and possible interaction between RGO and La3+@ZrO2 NF.

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Fig.1S. The PXRD of (a) La3+, (b) RGO, (c) ZrO2 NPs and (d) La3+@ZrO2 NPs / RGO.

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Fig. 2S. The SEM images of (a) RGO, (b) ZrO2 NPs/RGO, (c) 1% mol La3+@ZrO2/RGO, (d) 3% mol

La3+@ZrO2/RGO, (e) 5% mol La3+@ZrO2/RGO and (f) 6% mol La3+@ZrO2/RGO (3D rose like).

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Fig. 3S. The FTIR of (a) GO, (b) RGO, (c) La3+@ZrO2 NFs and (d) La3+@ZrO2 NFs/RGO.

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Fig. 4S. The EDX of La3+@ZrO2 NFs/RGO.

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Fig. 5S. Raman spectra of (a) GO, (b) RGO, (c) La3+@ZrO2 NFs /RGO and (d) La3+@ZrO2 NFs.

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Fig. 6S. (A) The CVs of La3+@ZrO2 NFs/RGO for 5 mM [Fe (CN)6]3/4- redox probe in 0.1 M KCl with

various scan rates (20-200 mV s-1) while (B) is a plot of square root of the scan rate (ν1/2) vs Ipc and Ipa.

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Fig.7S. DPV of 90×10-8 M SAL in phosphate buffer (pH=7.0) at (a) Bare GCE, (b) RGO/GCE,

(c) ZrO2 NPs/RGO/GCE and (d) La3+@ZrO2 NFs/RGO/GCE. DPV parameters were pulse height

of 0.035, step height of 0.05 V, pulse width of 0.020 V, pulse period of 0.3 s, and quiet time of 5

s that followed preconcentration time of 150 s.

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Fig.8S. The effect of pH on the electro-reduction of 20 μM SAL. Insets are relationships between pH vs.

Ipc and Epc.

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Fig. 9S. The effect of potential scan rate on the electro-reduction of 20 μM SAL. Insets are relationships

between scan rate (ν) vs. current, and log (ν) vs log Ipc.

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Scheme 2S. The proposed electro-reduction of SAL at La3+@ZrO2 NFs/RGO/GCE.

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Fig.10S. Selectivity of La3+@ZrO2 NFs/RGO/GCE toward 70×10-8 M SAL measurement in phosphate

buffer (pH 7.0). DPV parameters were pulse height of 0.035, step height of 0.05 V, pulse width of 0.020

V, pulse period of 0.3 s, and quiet time of 5 s that followed pre-concentration time of 150 s.

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Fig.11S. Mean plasma concentration of SAL measured by La3+@ZrO2 NFs/RGO/GCE (±SD) using DPV at

optimized conditions.

0 1 2 3 4 5 60

0.5

1

1.5

2

2.5

3

Time/hr

Con

cent

ratio

n/×1

0-8

M

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* Parameters are Cmax: maximum plasma concentration, tmax: Maximum plasma concentration,

t1/2: Elimination half‐life of SAL, AUC0−t=area under plasma concentration time curve from

time 0 to last time point with measurable drug concentration, AUC0−∞=area under plasma

concentration time curve extrapolated to infinity, Kel: Apparent terminal rate constant, CL:

Clearance, Vz=apparent volume of distribution in terminal elimination phase, Vss=volume of

distribution at steady state.

Table 1S. Pharmacokinetic parameters of SAL in rabbit plasma after oral administration

Parameters Measured values

Cmax (μg mL-1) 0.02± 0.001

Tmax (hrs) 2.2± 0.55

t½ (hrs) 3.02± 0.56

AUC 0-t(ng/mL h) 0.23± 0.02

AUC 0-∞(ng/mL h) 0.28± 0.03

Kel (h−1) 0.26± 0.01

CL (mL/min./kg) 800.4± 3.67

Vz (mg h/(ng/mL h) 1.18± 29.89

Vss (mg h/(ng/mL h) 1.09± 33.78