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Nanobioconjugated System Formed of Folic Acid-DeferrioxamineGa(III) on Gold Surface: Preparation, Characterization and Activities for Capturing of Mouse Breast Cancer Cells 4T1 Reza Karimi Shervedani, Fatemeh Yaghoobi, Mostafa Torabi, and Marzieh Samiei Foroushani J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b06066 • Publication Date (Web): 22 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Nanobioconjugated

System

Formed

of

Folic

Acid-Deferrioxamine-Ga(III) on Gold Surface: Preparation, Characterization and Activities for Capturing of Mouse Breast Cancer Cells 4T1

Reza Karimi Shervedani,* Fatemeh Yaghoobi, Mostafa Torabi, Marzieh Samiei Foroushani Department of Chemistry, University of Isfahan, Isfahan 81746-73441, I.R. IRAN

To whom correspondence should be addressed. Tel.: 98-31-37934922 Fax: 98-31-36689732

*

E-mail address: [email protected] (R. Karimi Shervedani)

1 ACS Paragon Plus Environment


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ABSTRACT: Folic acid-deferrioxamine B-gallium(III) system was assembled on the gold-mercaptopropionic acid surface through an effective method for the first time, and then, the assembled nanobioconjugated system, Au-MPA-FOA-DFO-Ga(III), was successfully tested for capturing of the mouse breast cancer cells 4T1. Physicochemical characteristics of the constructed system were studied by attenuated total reflectance Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, cyclic and differential pulse voltammetry, and electrochemical impedance spectroscopy (EIS). To evaluate the capturing ability of the folate receptor (FR) expressed cancer cells by the prepared system, the 4T1 cells were tested as a model of FR-expressed cells, and the human foreskin fibroblast cells as a model of no FR-expressed cells. The presence of cancer cells on the system surface was successfully detected by EIS based on variations of the charge transfer resistance (Rct) of the [Fe(CN)6]3/4 redox probe at the Au-MPA-FOA-DFO-Ga(III) electrode system/solution interface. Large variations observed in the Rct of the electrode, from 30.26 ± 0.04 to 227.50 ± 0.02 k, supported high affinity of the Au-MPA-FOA-DFO-Ga(III) system for 4T1 cells. Accumulation of the 4T1 cells onto the system surface was found to be time-dependent. The modified electrode exhibited rapid uptake kinetics for 4T1 cells with a t1/2 of 8.5 min. The experimental results are presented and discussed in this paper.


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1. INTRODUCTION Folic acid (FOA) conjugated with metal complexing agents are important systems regarding medical diagnosis and therapy treatment.1-3 Complexation of these conjugated systems with radioactive metal ions finally will lead to targeted radio-conjugated systems, which have been successfully used in preclinical imaging studies.4,5 The efficiency of synthesized system depends mainly on the nature of the (i) targeting agents, (ii) complexing molecule and (iii) metal ion.1 Thus, different types of these systems have been studied in the solution phase, and tested for pre-clinical and clinical applications,6,7 among them the system comprising of FOA function conjugated with deferrioxamine B-gallium ion complex, DFO-Ga(III), is especially important. Although several interesting papers have reported the chemistry and biochemistry of this system in solution phase,3,8 to the best of our knowledge, there has been no previous report about construction and characterization of FOA-DFO-Ga(III) bioconjugated system on gold surface. Thus, the characteristics of the designed system components; Gold, MPA, FOA, DFO and Ga(III) are briefly reviewed. Gold is a biocompatible metal9,10 that has been used in the treatment of nervous disorders,11 epilepsy,12 and as a contrast agent for diagnostic imaging and therapy purposes.13,14 Conjugating of the DFO-Ga(III) complex with FOA3,8 on gold surface can lead to construction of a new nanobioconjugated system that potentially benefits of the above-mentioned interesting properties. In addition, construction of the system on the solid surface allows excellent control over the chemistry and biochemistry of the designed structure.15-17 MPA is a biologically interesting thiol,18 with low molecular weight (LMW). The LMW thiols can be widely distributed in the tissues and cells.19 Monolayers of chemisorbed LMW



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thiocarboxylic acids are promising as appropriate platforms in the preparation of biocompatible surfaces.20,21 FOA, as a member of vitamin B family,22 is a popular molecule used for targeted drug delivery to tumor cells,23 due to high affinity of FOA towards folate receptors (FR) of the cancer cells.24 To date, many therapeutic and imaging agents, like NODAGA,1 DTPA and HYNIC,2 and DFO,2,3,8 have been successfully conjugated to FOA and their delivery to cancer cells have been studied via both in-vitro and in-vivo methods.1-3,25 DFO, a siderophore isolated from the actinobacter Streptomyces pilosus,26 has shown applications in (i) the chelation therapy of Fe(III) and Al(III) as an effective drug,27 (ii) medical imaging as a chelating agent,28 and (iii) cancer therapy as a therapeutic agent.29-34 Physicochemical behavior of the solid surfaces functionalized with DFO, directly35 or via mercaptopropionic acid (MPA),15,36 has been recently reported for different purposes by us. Ga(III) is the second metal ion after platinum, that has shown interesting behavior in cancer treatment. The complexed Ga(III) has shown a synergistic effect with other antitumor drugs.37 The similarity of Ga(III) with Fe(III) causes its interaction with cellular processes in the metabolism of Fe(III).38 This behavior has led to the development of Ga(III) complexes as antitumor and antimicrobial drugs and diagnostic agents.39 Conjugation of DFO-Ga(III) complex (as a theranostic agent) with FOA (as a targeting agent) has led to targeted conjugated structures with potential applications as a diagnostic agent in noninvasive imaging 3 as well as a therapeutic agent in cancer therapy.39 In spite of several papers dealing with medical applications of the bioconjugated FOA-DFO-Ga(III) in the solution phase,8 to the best of our knowledge, fabrication and


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characterization of the FOA-DFO and FOA-DFO-Ga(III) bioconjugated systems on the gold surface has not been reported yet. The

present

work

reports

design,

construction

and

study

of

the

Au-MPA-FOA-DFO-Ga(III) system in conjunction with cancer cells. Several techniques15-17 are used to characterize the system in each step, including attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), cyclic and differential pulse voltammetry (CV and DPV) and electrochemical impedance spectroscopy (EIS). To evaluate the capturing ability of the cancer cells by the characterized system, the mouse breast cancer cells 4T1, as a model of FR-expressed cell surface,40-42 are examined, in comparison with Human foreskin fibroblast (hFF) cells, as a model of no FR-expressed cells.43 The capturing process is traced by EIS method. The method is informative, efficient and nondestructive;17,44 measurements can be performed near the equilibrium potential which is especially interesting to transduce the signals of the biological events, carried out at the electrode/solution interface.45,46

2. METHODS AND MATERIALS 2.1. Materials and Reagents. Folic acid (FOA); (2S)-2-[[4-[(2-Amino-4-oxo-1Hpteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid, Ga(NO3)3.8H2O, deferrioxamine B (DFO) mesylate salt; N-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutanediamide, (dimethylamino)propyl)carbodiimide

(EDC),

n-hydroxysuccinimide

1-ethyl-3(3(NHS),

and

3-mercaptopropionic acid (MPA), were purchased from Sigma-Aldrich®. All other reagents were



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of analytical grade, purchased from commercial sources. Solutions were prepared with distilled water. The stock solution of 1.0 mM Ga(III) was prepared by dissolving a required amount of Ga(NO3)3.8H2O in 0.01 M HNO3 aqueous solution. The working solution, 10.0 µM Ga(III), was prepared by dilution of the stock solution. Acetic acid/acetate solution (AAS) was prepared by using 0.1 M acetic acid solution and the pH was adjusted using 0.1 M NaOH. Phosphate buffer solution (PBS), pH 7.4, was prepared by mixing desired amount of KH2PO4 and K2HPO4 salts in distilled water. The solution was placed in an autoclave under 100 °C for 24 h to sterile; then, it was cooled and stored at 4 °C. The 2.0 mM DFO solution was prepared by dissolving appropriate amount of its related salt in distilled water. Stock solution of 0.05 M FOA was prepared in 0.01 M NaOH, and then, the 0.1 mM FOA working solutions were prepared by dilution of the stock solution in the PBS, pH 5.5. 2.2. Cell Culture. The mouse breast cancer cells 4T1 samples were kindly provided by Isfahan University of Medical Sciences. The human foreskin fibroblast (hFF) cells were purchased from Royan Institute for Stem Cell Biology and Technology (RI-SCBT, Isfahan, I.R. IRAN(. The cell lines were received as suspended cells in the oxygenated sterile PBS, pH 7.4, and kept under oxygen till used. The mouse breast cancer cells 4T1 samples were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. The hFF cells were cultured in the same conditions, except high concentration of glucose was used in the culture medium.


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2.3. Apparatus. Electrochemical experimental setup; including instrumentation, measurements, acquisition and analysis of the data; was explained in our previous reports.15,47 Briefly, the electrochemical measurements were performed on a P/G Autolab 30 in a conventional three-electrode glass cell, using the modified gold electrode as working, a Pt rod with a large surface area as auxiliary electrode, and a saturated calomel electrode (SCE) as reference. All experiments were performed at room temperature. The potentials are reported vs. the reference electrode. The measured currents are normalized to the surface area of the related working electrode, resulting current densities (j) which can be easily compared. Differential pulse voltammetric measurements were performed at a step potential of 0.005 V, pulse height of 0.025 V, pulse width of 50 ms. The ATR-FTIR spectra of the gold electrode surface were obtained in the wavenumber range of 600 to 4000 cm1 by using Bruker Tensor 27 Spectrometer. The ATR-FTIR instrument is equipped with a CsI crystal (Pike Technologies). Electrodes are kept in a vacuumed desiccator for 15 min prior to obtaining the IR spectra. X-ray photoelectron spectroscopy (XPS) analysis was carried out on a VG Microtech Twin anode XR3E2 X-ray source and a concentric hemispherical analyzer operated at a base pressure of 5  1010 mbar using Al K (h  1486.6 eV). The XPS high resolution data were deconvoluted and fitted using PeakFit v4.12 software (235 Walnut St.S7, Framingham, MA 01702, USA), and the fitting results were plotted. 2.4. Preparation of Au-MPA-FOA-DFO-Ga(III) Electrode. The polycrystalline gold disk working electrode (0.0942 cm2 surface area, Azar electrode Co., Urmia, I.R. Iran) was cleaned first physically by polishing with aqueous slurries of alumina, and then,



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electrochemically in 0.5 M H2SO4, until reproducible voltammograms were obtained for redox reaction of AuO/Au on the Au surface.15,48 Cleaned electrode was immediately immersed in 20.0 mM MPA aqueous solution, sonicated for 2 min, and kept in this solution further for at least 12 h to form the Au-MPA SAM electrode. The modified electrode was washed with distilled water, and activated in 0.1 M PBS, pH 5.5, containing 2.0 mM EDC and 5.0 mM NHS for ~1.5 h,15 then the electrode was rinsed with distilled water and placed into 0.1 mM FOA solution, PBS, pH 5.5, for ~40 min (Please see Supporting Information, Figure S1) to fabricate Au-MPA-FOA modified electrode. The modified electrode was washed with distilled water, and again activated by EDC/NHS. Finally, the activated Au-MPA-FOA electrode was rinsed with distilled water, and immersed in 2.0 mM DFO aqueous solution for 30 min (Figure S2) at 20 °C to form Au-MPA-FOA-DFO modified electrode. Accumulation of Ga(III) onto the Au-MPA-FOA-DFO electrode was performed by immersion of the electrode into a 0.010 mM Ga(III) solution at pH 2.0 for 30 min35 to form Au-MPA-FOA-DFO-Ga(III) electrode. Step-by-step modifications of the gold electrode are illustrated schematically in Figure 1.


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Figure 1. Schematic illustration for step-by-step immobilization of FOA, DFO, and Ga(III) ion on Au-MPA electrode surface. Only a few of several possible structures is presented. For XPS measurements, a small part (~11 cm) of a gold recordable compact disk (CD-GR) was cut off and the protective layer of gold was removed by putting it in the concentrated (~65%) HNO3 for less than 2 min. Then, it was removed and washed with distilled water thoroughly. The unveiled gold was modified by MPA, FOA, DFO and Ga(III) by the above method, mentioned for polycrystalline gold disk. 2.5. Capturing of 4T1 Cells by Au-MPA-FOA-DFO-Ga(III) Electrode. To study the capturing ability of the constructed Au-MPA-FOA-DFO-Ga(III) electrode towards the 4T1 cells, one set of the electrodes was washed thoroughly with the sterile PBS, and immersed in sterile PBS containing 105 cells/ml 4T1 for an optimal time of 20 min (Figure S3). Then, the electrodes were removed, washed thoroughly with the sterile PBS, and tested for recording the EIS complex plane plots. Control Experiments. The Au-MPA-FOA-DFO-Ga(III) electrode was tested in the same way, but using hFF cells instead of 4T1 cells. Also, the Au-MPA-DFO-Ga(III) electrode (having no FOA) was prepared and tested for capturing the 4T1 cells.

3. RESULTS AND DISCUSSION 3.1. Construction and Characterization of the Nanobioconjugated System 3.1.1. Electrochemical Characterization. Electrochemical characterization was carried out first indirectly based on redox reaction of the [Fe(CN)6]3/4 probe at pH 3.0 (Figure 2), where FOA is in inactive mode, (Figure S4), and then, directly based on redox reaction of the



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immobilized

FOA

at

pH 8.0

(Figure 3),

where

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FOA

is

in

active

mode, (Figure S5, panels A & B). 3.1.1.1. Indirect Characterization Based on Inactive Mode of FOA. Step-by-step formation of the system on gold surface was also traced by monitoring the redox reaction of the negatively charged external redox probe, [Fe(CN)6]3/4, at pH 3.0 (Figure 2). Please note that the immobilized FOA is itself electrochemically inactive at pH 3.0 and in the applied potential window, 0.300 to +0.600 V (Figure S4). Formation of the MPA layer on Au surface has shown a blocking behavior against redox reaction of [Fe(CN)6]3/4 (Figure 2, curves a and b), leading to a decrease in the peak current (Ip), a potential shift to unfavorable direction, and an increase in the charge transfer resistance (Rct), which is of course a known effect.49 Likewise, we may expect the immobilized folic acid (Au-MPA-FOA) to impose a blocking effect against redox probe, however, an opposite effect is observed; the Ip is increased, peak separation (Epeak) and Rct are decreased (Figure 2, curve c). This effect is due to large electrostatic attraction (dominating effect) between highly protonated (positively charged) immobilized FOA at working pH 3.0 (pKa,surface of Au-MPA-FOA electrode is ~7.0, Figure S6),

with

negatively

charged

[Fe(CN)6]3/4.

Modification of Au-MPA-FOA surface with DFO and Ga(III) ions imparts further positive charge to the electrode surface,15,35 and thus, reinforces the electrostatic attraction effect, leading to further increase in Ip and decrease in Epeak, and Rct (Figure 2, curves d and e and Tables S1 and S2).

Clearly,

the

obtained

results

support

Au-MPA-FOA-DFO-Ga(III) electrode.


construction

of

the

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Figure 2.

(A) Cyclic and (B) differential pulse voltammograms obtained in 0.1 M AAS at

pH 3.0, in the presence of 0.5 mM [Fe(CN)6]3/4 on the (a) bare Au, (b) Au-MPA, (c) Au-MPA-FOA, (d) Au-MPA-FOA-DFO, and (e) Au-MPA-FOA-DFO-Ga(III) electrodes; scan rate: 100 mV s1. (C) The EIS complex plane plots obtained in the same conditions as (A) and (B), but at constant EDC = 0.200 V (vs. SCE) and in a wide frequency range from 10 kHz to 100 mHz, EAC = 5 mV superimposed on EDC. Symbols and trend lines show, respectively, the experimental data and the approximated (fitted) results based on model M1 (inset of panel (C)). The EIS complex plane plots for expanded scale at high frequency (low impedance region) are also shown as inset.



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3.1.1.2. Direct Characterization Based on Active Mode of FOA. To ascertain formation of the FOA, DFO, and Ga(III) modifying layers on the Au-MPA electrode surface, the behavior of the modified electrode is also investigated in the absence of any redox probe at pH 8 (Figure 3), where FOA can participate in direct electron transfer process at the electrode surface.50 While no significant faradaic reaction is observed for voltammograms obtained on the Au and Au-MPA electrodes (Figure 3, curves a and b), the Au-MPA-FOA electrode shows a well-defined voltammetric wave (Figure 3, curves c) with a formal potential of 0.040  0.008 V vs. SCE and n = 2, related to the direct electron transfer between immobilized FOA and the Au electrode base51,52 (Please see Supporting Information, Section 4, Figure S5 and Table S3).

The amount of FOA adsorbed on the Au-MPA (Γ) can be estimated via coulumetric charge (Q) consumed in the reduction reaction of the immobilized FOA (Figure 3, curves c, FOA,c = 6.80  1012 mole cm2). When DFO is immobilized onto the Au-MPA-FOA electrode surface, the faradaic peak current of FOA is significantly decreased (Figure 3, curve d, FOA,d = 2.41  1012 mole cm2), which, in turn, could be due to a decrease in the number of available FOA functions on the surface. Upon accumulation of Ga(III) on

the

Au-MPA-FOA-DFO electrode, the faradaic currents are further decreased (Figure 3, curve e, FOA,e = 0.71  1012 mole cm2). This behavior is due to tailoring of the surface functions by the Ga(III) ions,35 hindering redox reaction of the immobilized FOA. The obtained results support (i) the presence of the Ga(III) ions accumulated by the surface, and thus, construction of the Au-MPA-FOA-DFO-Ga(III) electrode, and (ii) remaining a fraction of the immobilized folate functions (FOA,e = 0.71  1012 mole cm2) active and available. This behavior, in turn, allowed successful targeting of the FRs-expressing 4T1 cancer cells.


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Figure 3.

(A) Cyclic and (B) differential pulse voltammograms obtained in 0.1 M AAS at

pH 8.0, on the (a) bare Au, (b) Au-MPA, (c) Au-MPA-FOA, (d) Au-MPA-FOA-DFO, and (e) Au-MPA-FOA-DFO-Ga(III) electrodes; scan rate: 150 mV s1. 3.1.2. Characterization by ATR-FTIR. The ATR–FTIR spectroscopy studies were used in conjunction with the results obtained by CV, DPV and EIS to support step-by-step attachment of MPA, FOA, DFO and Ga(III) modifying layers onto the gold surface (Figure 4). The gold surface shows a featureless behavior with trace effects of the gas phase H2O molecular clusters (curve a), while by adsorbing the MPA on gold, the characteristic peaks at 1417 cm1, 1520 cm1, 1683 cm−1 and between 2700 cm−1 and 3570 cm−1 appear. These peaks are attributed to the symmetric (s) and asymmetric (as) stretching frequencies of the carboxylate (COO) groups,53 stretching of C=O54 and stretching OH of MPA carboxylic acid function, respectively (curve b). The ATR-FTIR spectrum of the Au-MPA-FOA surface (curve c) shows multiple



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absorption bands in the range of 1600-1800 cm1, which can be assigned to the presence of amide (1600-1690 cm1) and carboxylate carbonyl (1690-1790 cm1) groups. Furthermore, the modifying layer is characterized with a band between 1490 and 1600 cm1 due to the coupling of N-H bending, and to some extent, to the C-N stretching, bands at 2850-3000 cm1 and 3000-3100 cm1 for alkane and aromatic C-H stretching vibrations and a broad band at ~660-1050 cm1 for N-H wagging character.55 Upon immobilization of DFO onto the Au-MPA-FOA surface (curve d), the intensity of the aromatic C-H peak decreases considerably as compared to that of the Au-MPA-FOA surface. This decrease in the band intensity is due to the deeper position of the C-H group in the Au-MPA-FOA-DFO layer that causes more loss of the incident and reflected IR rays. However, the intensity of CO and N-H absorption bands are not changed significantly because of the presence of these functional groups on DFO (Figure 1). Complexation of the Ga(III) ions with surface functional groups, expected to be essentially complexed with DFO functions, causes some changes in the vibrational frequencies of the modifying layer (curve e) including: (i) A significant decrease in the intensity of N-H bands in the ranges of ~660-1050 cm1, and 1490-1600 cm1. These behaviors can be due to deprotonation of the N-H bond. (ii) The complex formation allows more double-bond character in the C-N bond at ~1310-1400 cm1, in comparison with the free DFO, due to the resonance in the complex ring and consequently, a rise in C-N double-bond character (CN).56,57 These changes in bond lengths would shift the C-N stretching band to higher frequencies and increase its band intensity. This vibration band is very weak for free DFO (curve d). (iii) The band at 613 cm1 in the ATR-FTIR spectra of the Au-MPA-FOA-DFO-Ga(III) (curve e) can be


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attributed to the Ga-O stretching vibration.58 This band is absent in the ATR-FTIR spectra of the previous layers (Figure 4, curves a - d).

Figure 4.

ATR–FTIR spectra obtained on the (a) bare Au, (b) Au-MPA, (c) Au-MPA-FOA,

(d) Au-MPA-FOA-DFO, and (e) Au-MPA-FOA-DFO-Ga(III) electrode surfaces. 3.1.3. Accumulation of Ga(III) on Au-MPA-FOA-DFO Surface Traced by XPS. XPS is a powerful tool to identify all of the elements in periodical table and determine their oxidation



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state, except hydrogen and helium.59,60 Therefore, the presence of the Ga(III) ions on the system was further traced and approved by XPS (Figure 5). The XPS survey spectrum, obtained on the Au-MPA-FOA-DFO before (curve a) and after immersion in Ga(III) solution (curve b), is presented in Figure 5A. The spectrum indicates several peaks including C1s, S2p, N1s, and O1s related to the MPA-FOA-DFO system, and the Au2P, Au4P, Au4d and Au4f related to the Au substrate. The Ga(III) peak could be traced in survey spectrum around 1120 eV. Therefore, the high-resolution XPS measurements were performed in the range of 1100 to 1140 eV, where the peak appeared at ~1120 eV is attributed to Ga2p3/2 (Figure 5A, Inset).61-63 The obtained results support accumulation of the Ga(III) ions by the

modified

surface,

and

thus,

construction

of

the

Au-MPA-FOA-DFO-Ga(III)

nanobioconjugated system. To ascertain the role of DFO in accumulation (uptake and complexation) of Ga(III), the Au-MPA-FOA electrode (having no DFO) was prepared and immersed in Ga(III) solution in the same conditions. Then, the electrode was thoroughly washed and its survey and high-resolution XPS spectra were recorded (Figure 5B and its Inset). Since both spectra are featureless in the range of 1100 to1140 eV, it can be concluded that the immobilized FOA does not play essential role in accumulation of Ga(III) on the system while DFO is important for this purpose. The results obtained in section (3.1.1.2, Paragraph 2) revealed that a large fraction, 0.71 pmol cm2, of the FOA immobilized in the Au-MPA-FOA-DFO-Ga(III) system was active and achievable. This finding in conjunction with that obtained here, i.e. immobilized FOA does not accumulate the Ga(III) ion at the given conditions, is interesting regarding examination of the system for capturing of the FR-expressed 4T1 cancer cells.


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Figure 5.

Survey XPS spectra from 0 to 1200 eV obtained on (A) Au-MPA-FOA-DFO and

(B) Au-MPA-FOA electrodes; (a) before and (b) after immersion the electrodes in 0.010 mM Ga(III) solution at pH 2.0 for 30 min. The calibration is based on C1s peak at 284.7 eV. Insets show high-resolution XPS spectra of the electrodes surface in Ga2p3/2 region.



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Page 18 of 32

3.2. Capturing of the Mouse breast Cancer Cells 4T1 by the System. Interaction of the Au-MPA-FOA-DFO-Ga(III) system with FR-expressed cancer cells, as the second and most important part of this work, is traced by using the EIS.17,44-46 The measurements are carried out in the presence of [Fe(CN)6]3/4 external redox probe at the EDC = +0.200 V, where the FOA of the system is not electrochemically active (Figure 3, curve c). Accordingly, the nanobioconjugated system was immersed in the 4T1 cells solution for an optimal time of 20 min (Figure S3). Then, it was removed, washed with blank solution, and used for recording the EIS complex plane plots (Figure 6, curve f). For comparison, the EIS data obtained on the previous layers, formed during step-by-step construction of the system, are also presented (curves a - e).

Figure 6.

The EIS complex plane plots obtained in 0.1 M PBS, pH 7.4, in the presence of

0.5 mM [Fe(CN)6]3/4 on the (a) bare Au, (b) Au-MPA, (c) Au-MPA-FOA, (d) Au-MPA-FOA-DFO, (e) Au-MPA-FOA-DFO-Ga(III), and


Page 19 of 32

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(f) Au-MPA-FOA-DFO-Ga(III)-4T1 electrode systems at constant EDC = 0.200 V (vs. SCE) and in a wide frequency range from 10 kHz to 100 mHz, EAC = 5 mV superimposed on EDC. The EIS data were fitted into the appropriate equivalent circuit models, and the quantitative results were extracted. Two points should be explained here: (i) The capturing kinetics of the 4T1 cells obtained on the Au-MPA-FOA-DFO-Ga(III) electrode was found to be fast; for example, the time needs to achieve 50% of maximal electrode response (t1/2) is 8.5 min (Figure S3). This half-time may be compared with that reported for uptake of KB cell by FOA-DFO-Ga(III) in solution phase (3 min).8 (ii) A comparison between the quantitative results (Table 1, compare the Rcts as well as the Cdls), obtained by fitting the EIS data into models M1 & M2 (Figure 6, Insets), indicates that Rct is increased by a factor of ~8 upon condition of the system into the 4T1 cells solution, supporting effective accumulation of these cells onto the electrode surface. This behavior clearly implies the high affinity of the Au-MPA-FOA-DFO-Ga(III) system for FR-expressing 4T140-42 cancer cells.

Table 1. Electrochemical parameters extracted from EISs (Figure 6) obtained on Au, Au-MPA, Au-MPA-FOA, Au-MPA-FOA-DFO, Au-MPA-FOA-DFO-Ga(III), and Au-MPA-FOA-DFOGa(III)-4T1 electrodes in the presence of 0.50 mM [Fe(CN)6]3/4 in 0.1 M PBS, pH 7.4.a Electrodes Au

Rs /()

Rct /(k)

Cdl/(µF)

g

kapp/(cm s-1)  106

96.7  0.5

3.3  0.2

3.10  0.06

0.93

1.60  0.08

Au-MPA

157.7  0.4

71.42  0.02

1.05  0.01

0.89

0.080  0.008

Au-MPA-FOA

151.5  0.5

167.30  0.02

1.05  0.01

0.89

0.032  0.002

Au-MPA-FOA-DFO

142.6  0.4

102.31  0.02

1.05  0.01

0.89

0.055  0.003

Au-MPA-FOA-DFO-Ga(III)

94.7  0.7

30.26  0.04

1.26  0.02

0.96

0.18  0.04

Au-MPA-FOA-DFO-Ga(III)-4T1

91.4  0.9

227.50  0.02

1.05  0.01

0.94

0.023  0.002



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Page 20 of 32

Q = Cdlg [Rs1  Rct1]1g, kapp = RT/n2F2RctCA for redox system in solution phase; Rct is electron transfer resistance; n is number of transferred electron which is equal to one; C is concentration of redox probe (0.5 mM); F is faraday number (96,500 C mol−1); A is surface area of the electrode, 0.0942 cm2, and the R and T parameters have their usual meaning (R is gas constant 8.315 J mol−1 K−1 and T is Kelvin temperature). a

Several control experiments were performed to examine the selectivity of the Au-MPA-FOA-DFO-Ga(III) electrode system towards the FR-expressing cancer cells. Thus, the electrode was incubated in the sterile PBS at pH 7.4 containing hFF cells as a model of no FR-expressed cells.43 Then, the electrode was washed thoroughly with the sterile PBS, and its EIS

complex

plane

plot

was

recorded

in the presence of

[Fe(CN)6]3/4

probe

(Figure S7, panel A, compare curves a & b). Since the Rct is not significantly changed by this treatment, it

can

be

concluded

that

the

hFF

cells

have

not

associated

with

Au-MPA-FOA-DFO-Ga(III) system. Furthermore, a system with no FOA functions in its structure, Au-MPA-DFO-Ga(III), was prepared and examined in the same way (Figure S7, panel B) to verify the involvement of the cell surface FRs in mediating uptake of the 4T1 cells. According to the results, the Rct of the system did not significantly change also by this treatment, implying that neither the hFF nor the 4T1 cells have been associated significantly with the Au-MPA-DFO-Ga(III) system too. Thus, the control experiments support effective involvement of FOA in the uptake of 4T1 cells and selective behavior of the Au-MPA-FOA-DFO-Ga(III) system towards the tested FR-expressing cancer cells 4T1 (Figure 7).


Page 21 of 32

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Figure 7.

The histogram presenting charge transfer resistance obtained in 0.1 M PBS at

pH 7.4 in the presence of 0.5 mM [Fe(CN)6]3/4 on different bioconjugated modified surfaces.

4. CONCLUSION Molecular assemblies of folic acid/deferrioxamine/gallium(III) were constructed on the gold surface via a biocompatible linking thiol in the environmentally friendly conditions for the first time. Physicochemical properties of the layers, studied by CV, DPV, EIS, ATR-FTIR and XPS techniques, supported successful assembling of the layers, and thus, construction of the Au-MPA-FOA-DFO-Ga(III) nanobioconjugated system.



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Page 22 of 32

Overall, the ATR-FTIR and XPS surface studies in conjunction with the electrochemical results approved the presence of DFO-Ga(III) complex on the Au-MPA-FOA surface; still the voltammetric results revealed that the immobilized folate functions had remained active and available ( = 0.711012 mole cm2). This behavior, in turn, allowed successful targeting of the FRs-expressing 4T1 cancer cells. The presence of the 4T1 cells on the Au-MPA-FOA-DFO-Ga(III) electrode system was best traced by the EIS in the presence of [Fe(CN)6]3/4 as an external redox probe. The Rct was increased by a large amount upon immersion of the system into the 4T1 cancer cells solution. These results showed effective accumulation of the 4T1 cells on the electrode surface, and thus, approved high affinity of the Au-MPA-FOA-DFO-Ga(III) system for FR-expressing cancer cells. The control experiments did not reveal significant changes in the response of the electrodes for the following cases; (i) the Au-MPA-FOA-DFO-Ga(III) electrode towards hFF cells,

(ii) the

Au-MPA-DFO-Ga(III) electrode

towards

4T1

cancer

cells,

and (iii),

both of (i) and (ii) electrodes in the blank solution (did not contain 4T1 and hFF cells). Therefore,

it

was

concluded

that

(i) hFF

cells

had

not

been

associated

with

Au-MPA-FOA-DFO-Ga(III) system, and (ii) FOA, as the crucial component of the bioconjugated system, is active and achievable on the prepared system to capture selectively the FRs-expressing cancer cells. Therefore, the present study provides new insights into the design and development of nanomaterials as theranostic systems for cancer cells. In effect, it is proposed that the prepared Au-MPA-FOA-DFO-Ga(III) system be used in FOA and Ga(III)-related drug delivery and


Page 23 of 32

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diagnostic MRI imaging in the form of nanoparticles. This part of the research is in progress in our laboratory.

ASSOCIATED CONTENT Supporting Information. Cyclic voltammograms regarding the immobilization time of FOA and DFO, the redox behavior vs. pH of Au-MPA-FOA electrode, Tables S1 & S2 of EIS quantitative data for characterization of the studied system, Table S3 containing coulometric data & surface concentration, , of FOA; the EIS complex plane plots obtained for the capturing-time of 4T1 cells, and for control experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel: +98-31-37934922. Fax: +98-31-36689732. E-mail: [email protected].

ACKNOWLEDGMENT The authors gratefully acknowledge the University of Isfahan (UI) providing research facilities. The Iran National Science Foundation, Vise-Presidency for Science and Technology (INSF-VPST) is acknowledged for providing research facilities. Mr. Mohammad Keshtkar, Department of Medical Physics, Isfahan University of Medical Science (IUMS) is acknowledged for culturing mouse breast cancer cells 4T1.



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Page 24 of 32

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Table of Content (TOC)

FR

Au

4T1

Au

DFO Ga(III) FOA

MPA

Au

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Capturing response


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Nanobioconjugated System Formed of Folic Acid ... - ACS Publications

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