Combined Experimental and Computational Study of the In Situ

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

A Combined Experimental and Computational Study of the In Situ Adsorption of Piroxicam Anions on the Laser-Generated Gold Nanoparticles Roya Binaymotlagh, Hossein Farrokhpour, Hassan Hadadzadeh, Seyedeh Zohreh Mirahmadi-Zare, and Zahra Amirghofran J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b12962 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 50

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

The Journal of Physical Chemistry

127x108mm (95 x 95 DPI)

ACS Paragon Plus Environment


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

The Journal of Physical Chemistry C A Combined Experimental and Computational Study of the In Situ Adsorption of Piroxicam Anions on the Laser-Generated Gold Nanoparticles Roya Binaymotlagh,†,‡ Hossein Farrokhpour,*,† Hassan Hadadzadeh,*,† Seyede Zohreh Mirahmadi_Zare,‡ and Zahra Amirghofran§ †

‡

Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran

Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

§

Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Number of Pages: 49 Number of Figures: 10 Number of Table: 1

*Corresponding

authors,

Hassan Hadadzadeh Professor of Inorganic and Bioinorganic Chemistry Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran E-mail address: [email protected] Hossein Farrokhpour Associate Professor of Physical Chemistry Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran E-mail address: [email protected]

1 ACS Paragon Plus Environment

Page 2 of 50

Page 3 of 50

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


Abstract

In the present work, gold nanoparticles–piroxicam anion conjugates (Au NPs–Pir) were synthesized in a green method using laser ablation (LA) of a gold foil in an aqueous solution of anionic piroxicam (NaPir). The produced Au NPs–Pir conjugates were characterized by different spectroscopic techniques and transmission electron microscopy (TEM). The fluorescence and absorption spectra of Au NPs–Pir colloidal solution were recorded at different concentrations of the Au NPs to investigate the effect of conjugation on the spectral properties of both the free Pir anion and Au NPs. Comparing the FT-IR spectrum of the colloidal solution of Au NPs–Pir with that of the solution of the free Pir anion indicates that this anion binds to the surface of Au NPs through its different binding sites, viz., the pyridyl nitrogen atom, the SO2 group, and the amide oxygen atom. To support the experimental observations, theoretical calculations were also performed in order to evaluate the interaction of different binding sites of Pir with one Au atom and the effect of this interaction on the IR spectrum of Pir. In a more realistic theoretical study, the optimized geometry of Pir on the Au(111) surface was calculated and its orientation toward the surface of Au was obtained. The calculations show that the pyridyl ring of Pir prefers to orient perpendicular to the surface of Au, and the O atoms of the SO2 and amide groups are responsible for the interaction with the surface. The cytotoxic effect of the free Pir anion and Au NPs–Pir conjugates against the Jurkat T-cells was studied using an MTT assay. The results show that only the Au NPs–Pir conjugates have significant anticancer activity against the Jurkat Tcells with an IC50 value of 33.8 ± 2 μg/mL. In comparison with the pure Au NPs and free Pir, the Au NPs–Pir conjugates show a significant antimicrobial activity against two pathogens including Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus).



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

1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are medications with the analgesic and antipyretic effects.1,2 Studies in the field of cancer therapy have shown that NSAIDs have chemoprevention3,4 and chemosuppression5,6 effects and they can be good candidates against the development of solid tumors such as intestinal and bladder tumors.4 Oxicams are the most wellknown

members

of

NSAIDs.7

Piroxicam

(4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-

benzothiazine-3-carboxamide-1,1-dioxide) is currently the most widely used oxicam to treat arthritis in patients.7–10 Studies on piroxicam have shown its chemopreventive effects in different cancer cell lines5 such as lung, colorectal,11 and breast cancers.3,12 Although, it is well known that the majority of NSAIDs act as a cyclooxygenase inhibitor in their anti-inflammatory activities,13 their anticancer properties have not been clearly understood. It is supposed that they exhibit anticancer effects through both inhibition of protein level and/or transcriptional process.14 It is well known that the delivery way of a drug has an important role in its effectiveness.15 Developments in the field of nanotechnology have provided new tools for delivering drugs and gens16 such as carbon nanotubes,17 silica nanoparticles,18 gold nanoparticles,19 and quantum dots (QDs).20 Among these nanomaterials, Au NPs are excellent candidates for fabricating drug delivery vehicles.21–24 Ease of synthesis, inertness, nontoxic nature, and their ability to be functionalized with variety of drugs and ligands25 make them suitable tools for transporting and releasing of therapeutic drugs to a specific tissue.25 The most challenging problem of chemotherapy is the non-specific distribution of anticancer drugs which reducing the therapeutic dose within cancer cells. Therefore, to increase the efficiency of the medicinal treatment, a higher dose is required to achieve optimal cell kill. This high-dose is not safe and may damage healthy tissues (healthy cells) in addition to the cancer (target) cells. Nanoparticles provide a


Page 4 of 50

Page 5 of 50

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


controlled situation for transporting the therapeutic agents to the target site, leading to enhancement of the efficient concentration of the anticancer drug in the cancer cells, and hence, a lower dose is required to produce a desired therapeutic effect. Thus, their undesirable influence on normal tissues and also adverse unwanted side effects can be reduced.23 In recent years, many interesting studies have been performed about the application of functionalized Au NPs as the drug delivery vehicles.26 In 2012, Craig et al.27 used Au NPs and gold nanorods (Au NRs) as vehicles for delivering of Pt(IV) prodrugs. This combination enhanced the cellular uptake of the Pt drugs and reduced their cytotoxicity toward healthy tissues in comparison with free cisplatin. Aryal et al.24 conjugated doxorubicin (DOX) to the thiolated Au NPs through a pH-sensitive hydrazone bond. It was found that the Au NPs–DOX conjugates show more stability at physiological conditions than the free DOX molecules. The Au NPs–DOX conjugates are also localized at perinuclear regions which can enhance the cytotoxicity of DOX. In the presence of Au NP–BSA (BSA = bovine serum albumin) as a nanocarrier, Murawala et al.23 reported the cytotoxicity enhancement of the anticancer drug methotrexate (MTX) to the MCF–7 breast cancer cells. Their results have shown that the Au NPs–BSA bioconjugated system acts as a good carrier for MTX and has an ability to enhance its efficiency toward the target cells. In 2004, Tom and his co-workers28 investigated the adsorption of ciprofloxacin molecules on the surface of Au NPs. They showed that the NH moiety of the piperazine ring of ciprofloxacin can bind strongly to Au NPs. They also showed that the rate of the drug-releasing in a basic medium is faster than that in an acidic one. In 2014, Rodrigues et al.1 used a double layer of a gemini imidazolium-based amphiphile molecule to synthesize water-soluble gold nanoparticles. They loaded the piroxicam molecule as a poorly water-soluble drug on Au NPs and studied its bioactivity. In addition, they used a colorimetric detection method to measure the 4 ACS Paragon Plus Environment


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

peroxidase activity of cyclooxygenase (COX) through the formation of an oxidized form of N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) using the A549 p6 cell line. The problem of multi-drug resistant (MDR) bacteria is serious for human health.29 According to a report, published by the US Center for Disease Control and Prevention (CDC), the antibiotic-resistant bacteria cause millions of infections and thousands of deaths every year in the USA.30 The high surface-to-volume ratio of nanoparticles allows loading of abundant functional biomolecules, providing multivalency on the nanoparticle surface to increase their interactions. Conjugation of common antibiotics such as ciprofloxacin and vancomycin with NPs results in the enhancement of their antibacterial activity, and lower minimum inhibitory concentration (MIC) values are observed in comparison with free antibiotics.28,30,31 Generally, the synthesis of Au NPs is mainly performed using chemical methods.32 However, the chemical methods, beside their advantages, have some problems to use their final products (i.e. NPs) in biological systems due to the impurities resulting from the use of reducing agents, surfactants, chemical precursors, and other related chemicals.33 In the past decade, pulsed laser ablation in liquid (PLAL) has been shown to be a promising method to prepare NPs with different composition including nanometals, nanosemiconductors, carbon-based nanomaterials, and nanoalloys.34 The preparation of metallic NPs by PLAL has useful advantages including high biocompatibility, high stability, high purity, and high dispersibility.35 In the PLAL method, it is possible to generate nanoparticles directly from a solid target (metal target) without addition of chemical reagents. This single step method allows the generation of bare nanoparticles and their further conjugation with desired molecules such as drugs, ligands, and biomolecules. In this work, we used the PLAL method as a facile and green approach for direct conjugation of Pir on the surface of the laser-generated Au NPs (Au NPs–Pir) in a Pir solution. It is worthy to 5 ACS Paragon Plus Environment

Page 6 of 50

Page 7 of 50

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


mention that in this in situ generation of the Au NPs–Pir conjugates, the Pir anion is not a reducing agent or a chemical precursor to produce the Au NPs. Both absorption and fluorescence measurements were used to investigate the interaction of Pir with the Au NPs. The quantum chemical calculations were also employed to obtain information about the interaction, binding sites, structure, and orientation of Pir on the Au NPs surface. To the best of our knowledge, there is no computational work on the study of the relative orientation of Pir to the gold surface. Investigation of the structure and orientation of molecules adsorbed on the metal surface is an essential step for their future applications in biotechnology and nanomedicine.36–38 The anticancer effect of the Au NPs–Pir conjugates on the Jurkat T-cells was also studied. Finally, the antibacterial activity of the Au NPs–Pir was evaluated against Gram-negative Escherichia coli (ATCC 8739) and Gram-positive Staphylococcus aureus (ATCC 6538).

2. Experimental Section 2.1. Materials and methods The experimental setup is schematically shown in Scheme S1 (see Supporting Information). The main components of the system include a small beaker containing solution, a prism, and a convex lens (focal length = 100 mm) to control and focus the laser beam. The laser ablation was performed by focusing the first harmonic of Nd:YAG laser pulse (Quantel model TG-80, France, λ = 1064 nm, τ = 5 ns) perpendicularly aligned to the gold surface and operating at a repetition rate of 10 Hz, energy 360 mJ/pulse, and corresponding fluence of 10 J/cm2. The gold nanoparticles were prepared in situ using pulsed-laser ablation of an Au target (99.99% purity) in an aqueous solution of the piroxicam anion (NaPir, 10–4 M) (see Supporting Information, 6 ACS Paragon Plus Environment


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

Scheme S1). The piroxicam solution was continuously stirred during laser irradiation. After the preparation of the Au NPs–Pir colloidal solution, the sample was centrifuged (at 20000 rpm for 10 min) to obtain the precipitate of Au NPs–Pir conjugates. The supernatant was quantified for Pir by recording the absorption spectrum at 352 nm. The quantity of Pir adsorbed on the Au NPs was calculated indirectly by subtracting Pir concentration of the supernatant from the initial concentration of Pir (10–4 M) used for the conjugation. In addition, the quantity of Au in the precipitate was determined by inductively coupled plasma-optical emission spectroscopy (ICPOES) (Optima 7300 DV). The total concentration of Au NPs–Pir conjugates (in μg/mL) was calculated by adding the Au concentration (in μg/mL) to the quantity of Pir (in μg/mL) adsorbed on the Au NPs. Piroxicam, trypan blue, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), RPMI 1640 medium, phenol red, and glucose (C6H12O6 ≥ 99.5%) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Kentucky, USA). All the solutions were prepared in doubly distilled deionized water. All reagents were of analytical grade and were used as received without further purification. The spectral data were collected at the ambient temperature. The electronic absorption spectra of the free Pir and Au NPs–Pir were recorded on a JASCO 7580 UV–Vis–NIR doublebeam spectrophotometer using quartz cell with a path length of 10 mm. Fourier transform infrared spectra were recorded on an FT–IR JASCO 680-PLUS spectrometer in the region of 4000–400 cm–1 using KBr pellets. Steady state luminescence measurements were performed on a SHIMADZU RF 5301PC spectrofluorophotometer. The TEM images were obtained using a Zeiss-EM10C-80 kV (Germany). The size distribution of NPs was determined from the TEM images using the SPSS program. The laser parameters were the same for all experiments. 7 ACS Paragon Plus Environment

Page 8 of 50

Page 9 of 50

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


2.2. Cell culture and cytotoxicity assay The human lymphocyte Jurkat T-cells were obtained from the Iranian Cell Bank (Pasteur Institute of Iran, Tehran). The cancer cells were kept in RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin-streptomycin at a density of 1 × 106 cells/mL in a humidified 5% CO2 incubator at 37°C. The viability of Jurkat T-cells was determined by an MTT reduction assay in the presence and absence of the free Pir anion, Au NPs, and Au NPs– Pir. Briefly, 15 × 103 cells/well were seeded into a sterile 96-well plate (Nunclone, Thermo scientific CA, USA) and then treated with different concentrations (0.1, 1, 10, 25, 50, and 100 μg/mL) of each compound (Pir, Au NPs, and Au NPs–Pir) in a final volume of 100 μL. The negative control was the Jurkat T-cells without the compounds treatment and the positive control was Jurkat T-cells treated with cisplatin (50 μg/mL). After 24 h of incubation, 10 μL of the MTT solution (5 mg/mL in PBS) was added to each well and the cells were incubated again at 37°C for 4 h. After removing the medium from each well, the purple formazan crystals formed in the cells were dissolved by adding 150 μL of DMSO. The optical density (OD) of the dissolved formazan in the wells was measured with a microplate reader (Bio-Tek, Nevada, USA) at 570 nm with a background subtraction at 630 nm. The absorbance of formazan in the negative control cells was taken as 100% viability. The experiment was carried out in triplicate and repeated three times for each compound (Pir, Au NPs, and Au NPs–Pir). 2.3. Antibacterial test The standard strains of Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 6538) were obtained in a lyophilized form (purchased from the Pasture Institute of Iran). Luria-Bertani broth (LB, Sigma-Aldrich) was purchased as a powder and dissolved in water. The



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

Page 10 of 50

antibacterial activity of the Au NPs–Pir conjugates was determined based on a microwell dilution method.39 Briefly, the bacteria were cultured overnight at 37°C in an LB medium and adjusted to a final density of 106 CFU/mL (CFU = Colony Forming Unit). A 96-well plate was prepared by dispensing into each well 180 µL of the LB medium (containing phenol red (0.05%) and glucose (0.5%)), 10 µL of each compound (Pir, Au NPs, and Au NPs–Pir) with different concentrations (10–200 µg/mL), and 10 µL of the bacterial inoculum (at a final density of 5 × 104 CFU/mL in each well). The experiments also included two controls, viz., a positive control (the wells containing the bacterial inoculum, antibiotics (penicillin 1000 units/mL and streptomycin 1 mg/mL), and the LB media), and a negative control (the wells containing the bacterial inoculum and the LB media).39,40 The final volume in each well was 200 µL. Then, the 96-well plate was incubated at 37°C for 12 h. The color change of the phenol red indicator from red to yellow was due to the formation of an acidic waste by the growth of the microorganism. After incubation, the lowest concentration of the Au NPs–Pir, without an observable bacterial growth or a color change, was taken as the MIC value.39 The experiments were carried out in triplicate. 2.4. Computational details The different structures of Pir interacting with one Au atom (Au–Pir) were optimized using the density functional theory (DFT) method employing the CAM-B3LYP functional.41,42 The 6311+G(d,p) basis set was selected for the C, H, N, O, and S atoms and LANL2DZ was used for the Au atom. The interaction energy between the Au atom and different binding sites of Pir was calculated using the super-molecular approach considering the basis set superposition error (BSSE).43,44 The frequency calculations at the same level of theory were performed to obtain the IR spectra for different types of the Au–Pir complexes. In addition, to provide information about 9 ACS Paragon Plus Environment

Page 11 of 50

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


the orientation of Pir relative to the Au surface, the “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method

45

was employed. An Au(111) surface,

including two atomic layers of Au (containing 264 Au atoms), was selected and the Pir anion was placed horizontally on this surface. To construct the Au surface, the unit cell of gold with a cubic symmetry and lattice constant of alattice = 4.077 Å was optimized using the CAMB3LYP/LANL2DZ method. The lattice parameters of the unit cell after the optimization were a = b = c = 3.921 Å. The optimized unit cell was used to construct a two-layer Au(111) supercell. A two-layer ONIOM model was selected for the calculations, so that the first Au-layer interacting with Pir was considered in a quantum mechanics (QM)-layer and the other Au-layer was considered in the molecular mechanics (MM) region (see Supporting Information, Figure S1). The CAM-B3LYP along with the 6-311+G(d,p) basis set and LANL2DZ was selected for the QM part of the system, and the UFF force field was used for the MM part of the system. It should be noted that the Pir anion and the first Au-layer were considered in the QM region to provide a good description for the interaction between Pir and the Au surface, and a reasonable geometry for Pir relative to the Au surface. During the optimization, the positions of the Au atoms of the surface were considered rigid, while the geometry of the Pir anion was flexible. All calculations were performed using the Gaussian 09 quantum chemistry package.46

3. Results and Discussion 3.1. TEM study Figure 1 and Figure S2 (Supporting Information) show the TEM image and the size distribution histogram of a colloidal solution of Au NPs–Pir. (Figure 1) 10 ACS Paragon Plus Environment


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

Page 12 of 50

According to the histogram (Figure S2, Supporting Information), the spherical Au NPs have an average particle size of 9 nm with a relatively narrow size distribution range. Preparation of size-selected metal nanoparticles in aqueous solutions is of great significance in the chemistry and physics of nanomaterials.47 Generally, the pulsed-laser ablation of metal targets in pure water results in rather large NPs (20–300 nm) with a broad size distribution (50–300 nm) due to the post-ablation agglomeration of small NPs.48 It is well known that the presence of a suitable ligand dissolved in water can effectively stabilize and reduce the size of noble metal NPs produced by PLAL method.39 The ligand molecules interact with the surface of NPs and prevent from the further coalescence.47-49 In PLAL of a gold plate (target) in the aqueous solution of Pir, the essential feature of the particle formation can be explained by the dynamic formation mechanism suggested by Mafune and co-workers.50 After the laser ablation of the gold target, a dense cloud of the gold atoms is formed immediately over the laser spot of the gold target. In comparison to the interaction between Au and Pir (Au–Pir) or Au and water (Au–H2O), the Au–Au interatomic interaction is stronger. Thus, the Au atoms are aggregated after the generation and formed the primary Au NPs until the Au atoms in the close vicinity are almost consumed. A slow diffusion of Au atoms outside this specific region causes a slow particle growth rate.50 In competition, this slow growth rate is terminated by coating the surface of Au NPs with the Pir anions, which diffuse through the aqueous solution toward the Au NPs.49 It is well known that the type and the concentration of stabilizing ligands have relevant effects on the size distribution of metal NPs obtained by PLAL method.39,51,52 Pir is a small anionic ligand with suitable electron donor moieties (including nitrogen and oxygen atoms) for trapping the small Au NPs.


Page 13 of 50

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


3.2. Absorption spectra To investigate the effect of the in situ conjugation on the spectral properties of Pir and Au NPs, the UV–vis absorption spectra of the free Pir anion and Au NPs–Pir system were recorded (Figure 2).

(Figure 2)

The electronic spectrum of Pir shows four absorption bands at 208, 252, 286, and 352 nm. The absorption bands at 208 and 252 nm can be assigned to the π → π* and n → π* transitions, respectively.53 The shoulder band at 286 nm, attributed to the n → π* transition of the C=O group of the amide moiety in Pir,7 is shifted to shorter wavelengths as the Au NPs concentration increases, revealing the involvement of this group in the conjugation process.7,53–55 The band at 352 nm in the free Pir, assigned to the n → π* transition of the pyridyl nitrogen,2 is blue-shifted during the ablation time. The changes in the UV–vis spectra of the colloidal solution of Au NPs–Pir in comparison with the Pir solution can propose the donation of the lone electron pairs of the Oamide and Npyridyl atoms to the Au NPs surface (i.e., a charge transfer from Pir to the Au surface).55,56 In the visible region, the absorption spectra show the characteristic plasmon band of Au NPs around 520 nm, which is consistent with previous reports (Figure 2).35 This band does not show any significant change in its original wavelength, which indicates that the agglomeration of Au NPs can be avoided in the Au NPs–Pir conjugated colloid. This phenomenon can be attributed to the formation of a protective Pir layer on the Au NPs surface, which stabilizes the colloidal suspension.35 In addition, the stability of Au NPs–Pir colloidal solution was confirmed by UV–vis spectroscopy. Figure S3 (see Supporting Information) shows



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

Page 14 of 50

the electronic spectra of a freshly prepared Au NPs–Pir sample and this same sample after 60 days.

In comparison with the UV–vis spectrum of the freshly prepared Au NPs–Pir sample, no significant spectral changes are observed for the two months aged Au NPs–Pir sample. The winered color of the Au NPs–Pir colloidal solution also remains the same (Inset of Figure S3). The obtained visible color and the spectral results confirm that the synthesized Au NPs–Pir conjugates are highly stable. As can be seen in Figure 2, the intensity of absorption bands of Pir solution increases in the presence of increasing amount of Au NPs. It should be mentioned that the electronic states of Pir can be changed by the formation of Au NPs–Pir conjugates, which can result in increasing the intensity of the Pir absorption bands. 28,35 ,56 ,57 3.3. Fluorescence measurements The affinity of Pir for the Au NPs was determined using the fluorescence quenching method. Figure 3 shows the emission spectra of the free Pir anion and Au NPs–Pir conjugates. The spectra were recorded at different concentrations of Au NPs (0.0 to 1.1 × 10–8 M). It is worthy to mention that the [Pir]Initial/[Au]Maximum concentration ratio is about 9090 (10–4 M/1.1 × 10−8 M), which means that at concentration range of 0.0–1.1 × 10−8 M for Au NPs, the aqueous solution of Pir (10–4 M) can provide the conditions of the surface binding sites saturation. The amount of the Pir anion attached to the Au NPs (at concentration of 1.1 × 10−8 M) was determined by UV-vis spectroscopy. Approximately, 7.2% of the free Pir anions bind to the surface of the Au NPs at concentration of 1.1 × 10−8 M.

(Figure 3)


Page 15 of 50

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


Pir has an emission peak at 370 nm following excitation at 260 nm.2 The fluorescence intensity of the solution gradually decreases as the Au NPs concentration increases (Figure 3), which indicates that the Au NPs act as a quencher for the fluorescence of Pir. It is well known that when a fluorophore is directly adsorbed or covalently attached onto the surface of a metallic nanoparticle, its fluorescence is strongly quenched.30,39 Consequently, the remaining fluorescence results from the unbound fluorophores in the solution. The quenching process is mostly due to the surface energy transfer from Pir to Au NPs. The quenching process is not only caused by an increase in the non-radiative rate, but also by a drastic decrease in the radiative rate of the fluorophore.39,57,58 There are two quenching mechanisms including static quenching (which originates from the formation of a non-luminous complex between the ground-state fluorophores and the quenchers) and dynamic quenching (which originates from the collision of the excitedstate fluorophores and the quenchers).35,59 The fluorescence quenching spectra and quenching mechanism can be analyzed by Stern-Volmer60,61 and Lineweaver-Burk62–65 equations (eqs 1 and 2, respectively), which have been commonly used to describe a dynamic or static quenching.65 F0/F = 1 + Ksv[Q]

(1)

1/(F0 − F) = 1/F0 + 1/(KLBF0[Q])

(2)

Where F0 and F are the fluorescence intensities in the absence and presence of a quencher, respectively, [Q] is the concentration of the quencher (Au NPs), KLB and KSV are the static and Stern-Volmer quenching constants, respectively.60–67 According to previous reports, the plot of F0/F vs. [Q] (Stern–Volmer plot) is linear for both mechanisms (static and dynamic).60,61 While, a linear plot of (F0 − F)−1 vs. [Q]−1 (Lineweaver–Burk plot) is observed only for a static



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

Page 16 of 50

quenching.62–67 In addition, an ascending curvature of the Stern-Volmer plot indicates a combined quenching mechanism.63,66,67 It can be observed from Figure S4 (Supporting Information) that the interaction of Au NPs with Pir results in a linear Stern-Volmer plot at concentration range of 1.7 × 10−9 – 1.1 × 10−8 M for Au NPs, which supports the hypothesis that only one type of quenching mechanism occurs.60,61 In addition, the Lineweaver-Burk plot (Figure S5, Supporting Information) demonstrates that the plot of (F0−F)−1 vs. [Q]−1 is linear that shows the presence of a static quenching for the Au NPs–Pir system.63,66,67 Absorption spectroscopy can be used to distinguish between two quenching mechanisms. The changes observed in the absorption spectra (Figure 2) are in a good agreement with the static quenching hypothesis.56,68–70 For the dynamic quenching, the absorption spectra of the fluorophore is not changed and only the excited-state of the fluorophore is influenced by the quencher. While, for the static quenching, a conjugate is formed between the ground-state of the fluorophore and the quencher, which would influence the absorption spectra of the fluorophore. As can be seen from Figure 2, as the concentration of Au NPs increases, the intensity of the Pir absorption bands enhances with a blue-shift in the wavelength maxima. The absorption spectrum of Pir is changed due to the formation of ground-state Au NPs–Pir conjugates.56 Thus, it can be concluded that the fluorescence quenching mechanism of Pir in the presence of Au NPs is static.68–70 For the in situ conjugation of Au NPs with Pir, KSV was calculated to be 2.4 × 108 M–1, which indicates that the Au NPs generated in the aqueous solution have an extraordinary strong quenching ability (Supporting Information, Figure S4).60,61 A high value of KSV also indicates a strong association between the nanoparticles and the fluorophore and further substantiates the involvement of a static quenching mechanism for the Au NPs–Pir system.60,61,71,72 For this static 15 ACS Paragon Plus Environment

Page 17 of 50

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


quenching process, the fluorescence spectra of the Au NPs–Pir colloidal solution can be also analyzed using the modified Stern-Volmer equation (eq 3):56,64,65 F0/(F0 − F) = 1/faKa[Q] + 1/fa

(3)

Where fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant, which is analogous to the associative binding constant for the quencher-acceptor system.56 A plot of F0/(F0 − F) vs. [Q]−1, shown in Figure S6 of the Supporting Information, gives a straight line, where the value of Ka can be calculated from the ratio of the intercept and slope. The value of Ka was found to be 3.1 × 108 M–1. It is worthy to note that eq 3 is somewhat similar to eq 2.64,65 In addition, the free energy of adsorption (ΔG0) can be estimated using the Van’t Hoff equation (eq 4): ΔGo = −RT ln(K)

(4)

Where K is analogous to the effective quenching constant Ka at the same temperature (T), and R is the ideal gas constant (1.98 × 10–3 kcal/mol K).70 The value of ΔGo was found to be −11.54 kcal/mol, indicating a spontaneous process for the binding of Pir to Au NPs.56 The Au NPs with a protective layer of Pir show an emission maximum located at 765 nm following excitation at 510 nm (Figure 4), which is consistent with previous reports.73

(Figure 4)

In Figure 4, it is observed that by increasing the generation time, the fluorescence intensity of Au NPs increases. The enhancement of the fluorescence intensity can be affected by the size and concentration of Au NPs.74 According to the data obtained from the UV–vis spectra, due to the Pir coating, there is no significant variation in the particle size. At a constant particle 16 ACS Paragon Plus Environment


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

Page 18 of 50

size, with increasing the concentration of Au NPs, the intensity of the emission band increases, which is consistent with the changes observed for the surface plasmon band of Au NPs (Figure 2). 3.4. Experimental FT-IR spectra The IR spectroscopy is used to obtain the detailed information about the binding modes of Pir to the Au NP surface.75 To characterize the Au NPs–Pir interaction, we mainly focused our study only on the most typical vibrations that are characteristic of the binding mode of Pir.76 The recorded IR spectra of the free Pir anion and the colloidal solution of Au NPs–Pir are shown in Figure 5.

(Figure 5)

In the IR spectrum of the free Pir anion, the peaks located at 1630, 1576, 1529, 1434, 1350, and 1298 cm–1 are assigned to the stretching of the amidic carbonyl group (C13=O14), C=C and C=N bonds in the pyridyl ring, carbonyl group (C23=O27), C5–N11 bond, C23–C26 bond, and the asymmetric stretching of the SO2 group, respectively (See Figure 6 for the numbering of the atoms in Pir).

(Figure 6) Comparison of the IR spectrum of the free Pir anion with that of the Au NPs–Pir colloidal solution shows that the peaks related to the C13=O14 bond, pyridyl ring, and SO2 group are shifted to the lower wavenumbers. Thus, the interaction of Pir with the gold surface seems to implicate the amide group, pyridyl ring, and thiazinic moiety. The red-shift observed for the 17 ACS Paragon Plus Environment

Page 19 of 50

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


amidic C13=O14 group is more than the other two peaks, thus the interaction of the O atom of the amidic C13=O14 group with the Au surface is stronger than the other interacting sites. Since the FT-IR spectra don’t give us enough information regarding to the relative orientation of Pir on the Au surface, the computational methods were used to study the probable binding sites and relative orientations of Pir involved in its interaction with Au NPs. 3.5. Calculated IR spectra In order to investigate the binding mode of Pir to the Au surface, the IR spectra of the optimized structures of different types of the Au atom–Pir conjugates (shown in Figure 6) were calculated. In these structures, an Au atom is interacting with the most probable binding sites of Pir. It is worth noting that the comparison of the calculated IR spectra of the Au atom–Pir conjugates with the free Pir anion is a theoretical guide for obtaining information about the binding mode of Pir to the Au surface. The calculated IR spectrum of the free Pir anion along with the IR spectra of the Au atom–Pir conjugates (Figure 6, structures I-V), are shown in Figure 7. In addition, the most important bands in the calculated IR spectrum of each structure have been assigned.

(Figure 7)

It can be seen that the IR spectrum of each Au atom-Pir conjugate is different from the calculated IR spectrum of the free Pir anion. In the structures I, II, and III in Figure 6, the Au atom is interacting with the O atom of the carbonyl group (C23=O27), the N atom of the pyridyl ring, and the O atom of the amidic carbonyl group (C13=O14), respectively. Comparison of the IR spectrum of structure I with that of the free Pir anion shows that the stretching bands of 18 ACS Paragon Plus Environment


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

Page 20 of 50

C23=O27 and C23–C26 shift to lower wavenumbers. In structure II, the Au atom is mostly interacting with the N atom of the pyridyl ring. It is worthy to mention that, although the Au atom is interacting with the nitrogen atom of the pyridyl ring, the related bands do not show any shift compared to those of the free Pir anion. However, their intensities have been reduced. In structure III, the Au atom is mainly interacting with the O atom of the amidic C13=O14 group and a considerable shift to lower wavenumbers can be seen for the stretching band of C13=O14. In structure IV, the Au atom is interacting with the N11 atom of the amide group in Pir and there are no significant changes in its IR spectrum in comparison with the Pir spectrum. There is only a slight shift in the stretching frequency of the amidic C13=O14 group, even though the Au atom does not directly interact with the O14 atom. In structure V, the Au atom is interacting with the O atom of the SO2 group in Pir. The main difference between the IR spectrum of structure V with the spectrum of the free Pir anion is a slight shift to lower wavenumbers for the O=S=O vibration. Based on the above explanations, the changes observed in the stretching band of the amidic C13=O14 group can demonstrate the interaction of Pir with the surface of Au NPs through the O14 atom. Similarly, the frequency shift in the stretching mode of C23=O27 can be considered as a finger print to determine that the Pir anion can also interact with the surface of Au NPs through O27. The observed shift in the stretching frequency of C23–C26 can be another evidence to support that the Pir anion can interact with the Au NPs surface through its O27. The interaction of the Au atom with the N atom of the pyridyl ring does not provide a significant change in the IR spectrum of the adsorbed Pir anion compared to its free form. The change in the relative band intensity of C13=O14 compared to the pyridyl ring shows that the Pir anion can also interact with the surface of Au NPs through its pyridyl ring. Similarly, the Au-Pir interaction through N11 of the amide group slightly shifts the amidic C13=O14 stretching band to higher


Page 21 of 50

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


wavenumbers and considerably changes the band intensity of the pyridyl ring relative to C13=O14. The calculated IR spectrum of structure V is very similar to the Pir spectrum, only the asymmetric stretching of O=S=O slightly shifts to lower wavenumbers compared to the calculated IR spectrum of Pir. The differences between the experimental IR spectra of the Au NPs–Pir conjugates and free Pir (Figure 5) are consistent with the theoretical results. The main differences between the experimental spectra are as follows: (i) A considerable shift to lower wavenumbers (1630  1600 cm–1) can be seen for the stretching of the amidic C13=O14 group. This red-shift is also seen in the calculated IR spectrum of structure III. Thus, the Pir anion is interacting with the Au surface through its O14. (ii) The band intensities of C13=O14 bond and the pyridyl ring are nearly equal to its corresponding band in the free Pir spectrum, while their relative intensities change in the IR spectrum of Au NPs–Pir. The calculated IR spectra of structure II and IV show this behavior compared to the calculated spectrum of Pir, which means that Pir is also interacting with the Au surface through the N atom of the pyridyl ring and the N11 atom of its amide group. (iii) The asymmetric stretching of SO2 group slightly shifts to lower wavenumbers. This change can be seen in the calculated IR spectrum of structure V, where Pir is interacting with the Au atom through its SO2 moiety. It can be concluded that four atoms of Pir, including the O atom of C13=O14, the N atom of the pyridyl ring, the N11 atom of the amide group, and the O atom of the SO2 moiety, are involved in the interaction with the surface of Au NPs.



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

Page 22 of 50

3.6. Interaction energy of an Au atom with Pir As mentioned in the previous section, four sites (atoms) of Pir are involved in the interaction with the Au NPs. To compare the strength of the interaction of these sites with an Au atom (structures II, III, IV, and V in Figure 6), their interaction energies were calculated in the gas phase using eq 5. Eint = E(Au–Pir) – E(Au) – E(Pir)

(5)

Where E(Au–Pir), E(Au), and E(Pir) are the electronic energies of the Au-Pir dimer, the free Au atom, and the free Pir anion, respectively. The calculated interaction energies were also corrected for BSSE. The calculated interaction energies for the structures II, III, IV, and V are –10.92, –8.30, –3.68, and –5.37 kcal/mol, respectively. The calculated interaction energies show that the N atom of the pyridyl ring has the highest interaction with an Au atom compared to the other interacting sites. Due to the spatial orientation of the lone electron pair on the N atom of the pyridyl ring, the pyridyl ring should be vertical to the Au surface to have the strongest interaction. In addition, the  electrons on the pyridyl ring can be another source for the interaction of Pir with the Au atom, however the interaction of the pyridyl N atom with Au is stronger than the  electrons. 3.7. The geometry of Pir on the Au surface Although the performed calculations show that Pir is mostly interacting with Au NPs through its O and N atoms of the amidic C13=O14 group and pyridyl ring, they do not provide information about the relative orientation and the changes in the geometry of Pir upon adsorption to Au. For this purpose, a two-layer of Au(111) surface was selected and the structure of Pir was optimized on the surface using the ONIOM method (Figure S1, Supporting Information). Figure 8 shows the optimized structure of Pir on the Au(111) surface.


Page 23 of 50

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


(Figure 8)

At the start of optimization, Pir was placed horizontally on the Au surface. It can be seen that the optimized geometry of the adsorbed Pir is considerably different from its free form. As can be seen in Figure 8, the pyridyl ring is perpendicular to the Au surface, which has been predicted in the previous section. Also, the two O atoms of C13=O14 and S=O bonds are oriented toward the Au surface and have direct interaction with the surface. As can be seen in Figure 8, there is a significant change in the structure of Pir due to the adsorption on the Au surface. Figure 9 shows the juxtaposition of the optimized structure of free Pir on its deformed structure (D–Pir) after the adsorption on the Au surface.

(Figure 9)

It can be seen that there is a considerable deformation in the structure of Pir upon adsorption on Au. The main change in the structure of Pir is related to the two angles in its skeleton. The value of C5–N11–C13 (Figure 6) in the free Pir anion is equal to 130.76, which decreases to 112.35 in the deformed Pir (D–Pir). The dihedral angle N6–C5–N11–C13 is 3.65 in the free Pir, which increases to 119.86 in D–Pir. This considerable change in the dihedral angle is related to the rotation of the pyridyl ring to occupy a vertical position on the Au surface. Recently, Hernandez et al.36 have studied the Raman spectra of the anionic Pir adsorbed on the 22 ACS Paragon Plus Environment


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

Page 24 of 50

silver and gold nanoparticles in DMSO/water solutions at different pHs. They reported that the pyridyl ring of Pir is closer to the metal atoms (Au or Ag) and is perpendicularly oriented to the metal surface in DMSO/water solution (pH ≥ 7). However, there is no evidence regarding to the participation of the (C=O)amide and S=O groups in the Pir-Au NPs interaction in their paper. Our results reveal that the (C=O)amide, S=O, and pyridyl moieties can simultaneously take part in the interaction of Pir with the Au NPs in an aqueous solution. It should be mentioned that the ONIOM calculations for the Au surface with more than two atomic layers has not significant effect on the geometry of the adsorbed Pir on the surface. The reason for this can be attributed to the distance between the atomic layers of the Au surface. The distance between two subsequent atomic layers of the Au surface is about 2.9 Å. Therefore, the distance between the third atomic layer and the first atomic layer is about 5.8 Å in a threelayer Au surface. As can be seen in Figure 8, the minimum distance between the Pir anion and the Au surface is about 2.37 Å (the vertical distance between the N atom of the pyridyl ring and the Au surface). Therefore, the distances between the interacting sites of the Pir anion with the second and third atomic layers are about 5.4 and 8.2 Å, respectively. As can be seen, the interaction of the Pir anion with the second atomic layer is very small. In addition, this interaction tends to zero in the third atomic layer of the Au surface. Therefore, the effect of the third atomic layer of the Au surface on the Pir anion is negligible and the first atomic layer has the main role in the adsorption mode of the Pir anion. 3.8. MTT cell viability assay The influence of the Au NPs, Pir, and Au NPs–Pir on the proliferation of the Jurkat Tcells was examined using the MTT assay. Figure 10 shows the plot of the inhibition percentage


Page 25 of 50

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


vs. the concentration of each compound, including Au NPs (0.1–100 μg/mL), Pir (0.1–100 μg/mL), and Au NPs–Pir (total concentration of 0.1–100 μg/mL, i.e.; containing 0.004–4 μg/mL of the adsorbed Pir and 0.096–96 μg/mL of the Au NPs).

(Figure 10)

The results show that the cell viability is strongly decreased with increasing the concentration of Au NPs–Pir and has a concentration-dependent manner. The IC50 values (the concentration of a compound to inhibit 50-percent cell growth) of the compounds are shown in Table 1.

(Table 1)

The results indicate that at a total concentration of 33.8 ± 2 μg/mL, the Au NPs–Pir conjugates (containing 32.4 μg/mL of Au NPs and 1.4 μg/mL of Pir) can decrease the viability of the Jurkat T-cells to 50%. Interestingly, the data show that at concentration of 0.1–100 μg/mL, the pure Au NPs and the free Pir have low inherent cytotoxicity to the Jurkat T-cells and it is estimated that at concentrations more than 200 μg/mL, both compounds may show a significant cytotoxicity. The MTT assay reveals that the adsorbed Pir is significantly more cytotoxic than its free form toward the Jurkat T-cells. A low cytotoxicity regarding to the free Pir may be due to this fact that it is poorly taken up by the cancer cells. Due to the pumping proteins on the cell surface, some drugs may be pumped out.77,78 By this way, the effective concentration of the drug reduces in the cells. Au NPs as safe, biocompatible, and low cytotoxic nanocarriers can influence



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

Page 26 of 50

the drug-cell interactions and enhance the therapeutic effects.23,24 Thus, it can be inferred that the loading of Pir on the Au NPs could significantly enhance the permeability and effective concentration of Pir in the Jurkat T-cells. 3.9. Antibacterial activity of Au NPs–Pir Progresses on the antimicrobial activity of Au NPs have been summarized in a recent review article.79 There are conflicting results on the antibacterial activity of the Au NPs. Generally, the Au NPs are not antibacterial, or have weak antibacterial activity at high concentrations. However, the antibacterial activity of the Au NPs colloidal solutions is possibly due to the bactericidal effect of co-existing chemicals in the bulk solutions. These co-existing chemicals are gold ions, surface coating agents, and chemicals used in the chemical synthesis of NPs, which are not completely removed from the Au NPs colloidal solutions (final products). However, it is well known that the Au NPs can be used as antibiotic carriers or delivery vehicles. 79,80 Conjugation of antibiotics such as ciprofloxacin and vancomycin with Au NPs significantly increases their bactericidal effect. The antibacterial activity of Au NPs–Pir was analyzed using the MIC measurements. MIC is defined as the lowest concentration of antibiotics or other related chemical substances at which there is no visible growth of microorganism (more than 99.9% lethality).81,82 Various concentrations of Au NPs (10–200 µg/mL), Pir (10–200 µg/mL), and Au NPs–Pir (total concentration of 10–200 μg/mL, i.e.; containing 0.4–8 μg/mL of the adsorbed Pir and 9.6–192 μg/mL of the Au NPs) were incubated with E. coli and S. aureus in an aqueous LB media. The bacterial growth was measured by visual inspection based on the color change of the phenol red indicator. Our results show that at the total concentration of 100 and 70 μg/mL, the Au NPs–Pir conjugates can inhibit the growth of E. coli and S. aureus, respectively, while the free Pir anion does not demonstrate a significant antibacterial activity in the range of 10–200 25 ACS Paragon Plus Environment

Page 27 of 50

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


μg/mL. Also, the pure Au NPs only begin to show inhibition effect at very high concentration of 200 μg/mL. At this very high concentration (200 μg/mL), a large number of Au NPs can directly interact with pathogens to cause some destructive effects.79 Previous studies have shown that NSAIDs have antibacterial activity due to their ability to inhibit the DNA synthesis or to impair the membrane activity.83 Compared with the Pir alone, the conjugation of Pir with Au NPs can significantly increase the permeability and cellular uptake of Pir. The MIC value of Au NPs–Pir against E. coli and S. aureus were found to be 100 (containing 4 μg/mL of the adsorbed Pir and 96 μg/mL of the Au NPs) and 70 µg/mL (containing 2.4 μg/mL of the adsorbed Pir and 67.6 μg/mL of the Au NPs), respectively. At the total concentration of 100 and 70 μg/mL of the Au NPs–Pir conjugates, the quantities of the Au NPs are 96 and 67.6 μg/mL, respectively. At these concentrations, the Au NPs cannot show inhibition effect. Thus, the antibacterial activity of the Au NPs–Pir conjugates is mainly due to the high localized concentration of Pir upon its release inside the bacterial cells, and the Au NPs can only act as a drug carrier and a concentrator. Thus, the antibacterial mechanism of the Au NPs–Pir conjugates is similar to that of the free Pir. We suggest that the difference between the antibacterial activity of the free Pir and the Au NPs–Pir conjugates is due to different cellular uptake. 4. Conclusion In this study, pulsed-laser ablation as a green physical method was used for the generation of Au NPs–Pir conjugates using IR (wavelength = 1064 nm) laser pulse with nanosecond-duration time. The Au nanoparticles were characterized by UV-vis, fluorescence spectroscopy and TEM. The TEM images showed that Pir can effectively control the growth processes of the Au NPs, which is consistent with the mechanism of the nanoparticle generation using the PLAL method. The UV–vis results show that the size and concentration of the Au NPs 26 ACS Paragon Plus Environment


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

Page 28 of 50

can be controlled by the in situ generation of the nanoparticles in the Pir solution. The fluorescence quenching method was employed to study the binding affinity of Pir to Au NPs. The results show that the Au NPs strongly interact with Pir through the formation of a groundstate complex. The theoretical calculations were also employed to interpret the effect of the PirAu NPs interaction on the IR spectrum of Pir, to determine the important interacting sites (atoms) of Pir involved in the interaction with the surface of the Au NPs, and to specify the possible configurations of Pir relative to the Au surface. Our findings show that the pyridyl ring of Pir is perpendicular to the Au surface and the most important interacting sites of Pir are the N atom of the pyridyl ring, the O atom of the amidic C13=O14 group, and the O atom of the SO2 group. Finally, our results show that the Au nanoparticles enhance the antibacterial activity of Pir against the E. coli and S. aureus bacterial cells. In addition, the Au NPs–Pir conjugates have significant cytotoxic activity against the Jurkat T-cells. Supporting Information The general schematic experimental setup used for the preparation of the conjugated Au NPs; the two-layer ONIOM model for the optimization of Pir on the Au(111) surface; size distribution histogram of the Au NPs prepared in Pir aqueous solution; absorption spectra of a freshly prepared Au NPs–Pir sample and this same sample after 60 days; the Stern-Volmer plot of the Au NPs–Pir; the Lineweaver-Burk plot obtained from the fluorescence spectra; the modified Stern-Volmer plot for the interaction of Pir with the Au NPs. Acknowledgements We would like to express our appreciation to the Isfahan University of Technology (IUT-Iran) and Royan Institute for Biotechnology for their generous support.


Page 29 of 50

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


References (1)

Rodrigues, M.; Calpena, A. C.; Amabilino, D. B.; Ramos-Lopez, D.; de Lapuente, J.; Perez-

Garcia, L. Water-soluble gold nanoparticles based on imidazolium gemini amphiphiles incorporating piroxicam. RSC Adv. 2014, 4, 9279-9287. (2)

Hadadzadeh, H.; Salimi, M.; Weil, M.; Jannesari, Z.; Darabi, F.; Abdi, K.; Khalaji, A. D.;

Sardari, S.; Ahangari, R. The piroxicam complex of copper(II), trans-[Cu(Pir)2(THF)2], and its interaction with DNA. J. Mol. Struct. 2012, 1022, 172-180. (3)

Sporn, M. B.; Suh, N. Chemoprevention of cancer. Carcinogenesis 2000, 21, 525-530.

(4)

Ritland, S. R.; Gendler, S. J. Chemoprevention of intestinal adenomas in the ApcMin mouse by

piroxicam: kinetics, strain effects and resistance to chemosuppression. Carcinogenesis 1999, 20, 51-58. (5)

Grossman, E. M.; Longo, W. E.; Panesar, N.; Mazuski, J. E.; Kaminski, D. L. The role of

cyclooxygenase enzymes in the growth of human gall bladder cancer cells. Carcinogenesis 2000, 21, 1403-1409. (6)

Goldman, A. P.; Williams, C. S.; Sheng, H.; Lamps, L. W.; Williams, V. P.; Pairet, M.; Morrow,

J. D.; DuBois, R. N. Meloxicam inhibits the growth of colorectal cancer cells. Carcinogenesis 1998, 19, 2195-2199. (7)

Darabi, F.; Hadadzadeh, H.; Ebrahimi, M.; Khayamian, T.; Rudbari, H. A. The piroxicam

complex of cobalt(II): synthesis in two different ionic liquids, structure, DNA-and BSA interaction and molecular modeling. Inorg. Chim. Acta 2014, 409, 379-389. (8)

Weon Cho, C.; Chul Shin, S. Enhanced controlled transdermal delivery of mexazolam using

ethylene-vinyl acetate. Iran. J. Pharm. Res. 2012, 11, 3-12. (9)

Cheong, H.-A.; Choi, H.-K. Enhanced percutaneous absorption of piroxicam via salt formation

with ethanolamines. Pharm. Res. 2002, 19, 1375-1380. (10)

Knapp, D. W.; Richardson, R. C.; Chan, T. C. K.; Bottoms, G. D.; Widmer, W. R.; DeNicola, D.

B.; Teclaw, R.; Bonney, P. L.; Kuczek, T. Piroxicam therapy in 34 dogs with transitional cell carcinoma of the urinary bladder. J. Vet. Intern. Med. 1994, 8, 273-278. 28 ACS Paragon Plus Environment


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

(11)

Page 30 of 50

Giardina, C.; Inan, M. S. Nonsteroidal anti-inflammatory drugs, short-chain fatty acids, and

reactive oxygen metabolism in human colorectal cancer cells. Biochim. Biophys. Acta, Mol. Cell Res. 1998, 1401, 277-288. (12)

Harris, R. E.; Alshafie, G. A.; Abou-Issa, H.; Seibert, K. Chemoprevention of breast cancer in

rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res. 2000, 60, 2101-2103. (13)

Knottenbelt, C.; Chambers, G.; Gault, E.; Argyle, D. J. The in vitro effects of piroxicam and

meloxicam on canine cell lines. J. Small Anim. Pract. 2006, 47, 14-20. (14)

Roy, S.; Banerjee, R.; Sarkar, M. Direct binding of Cu(II)-complexes of oxicam NSAIDs with

DNA backbone. J. Inorg. Biochem. 2006, 100, 1320-1331. (15)

Elbialy, N. S.; Fathy, M. M.; Khalil, W. M. Doxorubicin loaded magnetic gold nanoparticles for

in vivo targeted drug delivery. Int. J. Pharm. 2015, 490, 190-199. (16)

Sanderson, B. J. S.; Lam, R.; Alharthi, J.; Shapter, J. The potential of gold nanoparticle

conjugates to kill cancer cells in culture. Procedia Eng. 2014, 92, 26-29. (17)

Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q.; Chen, X.; Dai, H. Drug delivery with carbon

nanotubes for in vivo cancer treatment. Cancer Res. 2008, 68, 6652-6660. (18)

Jang, H. R.; Oh, H.-J.; Kim, J.-H.; Jung, K. Y. Synthesis of mesoporous spherical silica via spray

pyrolysis: Pore size control and evaluation of performance in paclitaxel pre-purification. Microporous Mesoporous Mater. 2013, 165, 219-227. (19)

Daniel, M.-C.; Astruc, D. Gold nanoparticles: Assembly, supramolecular chemistry, quantum-

size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 2004, 104, 293-346. (20)

Chakravarthy, K. V.; Davidson, B. A.; Helinski, J. D.; Ding, H.; Law, W.-C.; Yong, K.-T.;

Prasad, P. N.; Knight, P. R. Doxorubicin-conjugated quantum dots to target alveolar macrophages and inflammation. Nanomedicine: NBM 2011, 7, 88-96.


Page 31 of 50

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


(21)

Avvakumova, S.; Scari, G.; Porta, F. Au-thymine, thymidine and thymidine 5[prime or minute]-

monophosphate nanoparticles: chemical characterisation and cellular uptake studies into U87 cancer cells. RSC Adv. 2012, 2, 3658-3661. (22)

Juan-Juan, L.; Ying-Ying, Z.; Jun, W.; Ya, D. Gold nanoparticle-based drug delivery platform for

antineoplastic chemotherapy. Curr. Drug Metab. 2014, 15, 620-631. (23)

Murawala, P.; Tirmale, A.; Shiras, A.; Prasad, B. L. V. In situ synthesized BSA capped gold

nanoparticles: Effective carrier of anticancer drug Methotrexate to MCF-7 breast cancer cells. Mater. Sci. Eng., C 2014, 34, 158-167. (24)

Aryal, S.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Doxorubicin conjugated gold

nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J. Mater. Chem. 2009, 19, 7879-7884. (25)

Manson, J.; Kumar, D.; Meenan, B.; Dixon, D. Polyethylene glycol functionalized gold

nanoparticles: the influence of capping density on stability in various media. Gold Bull. 2011, 44, 99-105. (26)

Coelho, S. C.; Rangel, M.; Pereira, M. C.; Coelho, M. A. N.; Ivanova, G. Structural

characterization of functionalized gold nanoparticles for drug delivery in cancer therapy: a NMR based approach. Phys. Chem. Chem. Phys. 2015, 17, 18971-18979. (27)

Craig, G. E.; Brown, S. D.; Lamprou, D. A.; Graham, D.; Wheate, N. J. Cisplatin-tethered gold

nanoparticles that exhibit enhanced reproducibility, drug loading, and stability: a step closer to pharmaceutical approval? Inorg. Chem. 2012, 51, 3490-3497. (28)

Tom, R. T.; Suryanarayanan, V.; Reddy, P. G.; Baskaran, S.; Pradeep, T. Ciprofloxacin-protected

gold nanoparticles. Langmuir 2004, 20, 1909-1914. (29)

Neu, H. C. The crisis in antibiotic resistance. Science 1992, 257, 1064-1073.

(30)

Li, X.; Robinson, S. M.; Gupta, A.; Saha, K.; Jiang, Z.; Moyano, D. F.; Sahar, A.; Riley, M. A.;

Rotello, V. M. Functional gold nanoparticles as potent antimicrobial agents against multi-drug resistant bacteria. ACS nano 2014, 8, 10682-10686.



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

(31)

Page 32 of 50

Gu, H.; Ho, P.; Tong, E.; Wang, L.; Xu, B. Presenting vancomycin on nanoparticles to enhance

antimicrobial activities. Nano Lett. 2003, 3, 1261-1263. (32)

Petersen, S.; Barcikowski, S. In situ bioconjugation: Single step approach to tailored

nanoparticle-bioconjugates by ultrashort pulsed laser ablation. Adv. Funct. Mater. 2009, 19, 1167-1172. (33)

Warner, J. C.; Cannon, A. S.; Dye, K. M. Green chemistry. Environ Impact Assess Rev. 2004, 24,

775-799. (34)

Cristoforetti, G.; Pitzalis, E.; Spiniello, R.; Ishak, R.; Muniz-Miranda, M. Production of

palladium nanoparticles by pulsed laser ablation in water and their characterization. J. Phys. Chem. C 2010, 115, 5073-5083. (35)

Binaymotlagh, R.; Hadadzadeh, H.; Farrokhpour, H.; Haghighi, F. H.; Abyar, F.; Mirahmadi-

Zare, S. Z. In situ generation of the gold nanoparticles–bovine serum albumin (AuNPs–BSA) bioconjugated system using pulsed-laser ablation (PLA). Mater. Chem. Phys. 2016, 177, 360-370. (36)

Hernandez, M.; Corda, E.; Garcia‐Ramos, J.; Domingo, C.; Sevilla, P. SERS of the anti‐

inflammatory drug piroxicam adsorbed on the surface of silver or gold colloids as nanocarrier model. J. Raman Spectrosc. 2015, 47, 402-407. (37)

Chen, T.; Pal, A.; Gao, J.; Han, Y.; Chen, H.; Sukhishvili, S.; Du, H.; Podkolzin, S. G.

Identification of vertical and horizontal configurations for BPE adsorption on silver surfaces. J. Phys. Chem. C 2015, 119, 24475-24488. (38)

Mollenhauer, D.; Gaston, N.; Voloshina, E.; Paulus, B. Interaction of pyridine derivatives with a

gold (111) surface as a model for adsorption to large nanoparticles. J. Phys. Chem. C 2013, 117, 44704479. (39)

Hajareh Haghighi, F.; Hadadzadeh, H.; Farrokhpour, H. Investigation of the in situ generation of

oxide-free copper nanoparticles using pulsed-laser ablation of bulk copper in aqueous solutions of the DNA bases. RSC Adv. 2016, 6, 109885-109896. (40)

Agnihotri, S.; Mukherji, S.; Mukherji, S. Size-controlled silver nanoparticles synthesized over the

range 5-100 nm using the same protocol and their antibacterial efficacy. RSC Adv. 2014, 4, 3974-3983. 31 ACS Paragon Plus Environment

Page 33 of 50

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


(41)

Yanai, T.; Tew, D. P.; Handy, N. C. A new hybrid exchange–correlation functional using the

Coulomb-attenuating method (CAM-B3LYP). Chem. Phys. Lett. 2004, 393, 51-57. (42)

Dabbagh, H. A.; Shahraki, M.; Farrokhpour, H. Theoretical investigation of the borazine-

melamine polymer as a novel candidate for hydrogen storage applications. Phys. Chem. Chem. Phys. 2014, 16, 10519-10530. (43)

Boys, S. F.; Bernardi, F. d. The calculation of small molecular interactions by the differences of

separate total energies. Some procedures with reduced errors. Mol. Phys. 1970, 19, 553-566. (44)

Farrokhpour, H.; Karachi, S.; Chermahini, A. N. Theoretical modeling of the chirality

discrimination of enantiomers by nanotubular cyclic peptides using gas-phase photoelectron spectroscopy: An ONIOM spectroscopic calculations. J. Phys. Chem. A 2016, 120, 6780-6791. (45)

Dapprich, S.; Komáromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J. A new ONIOM

implementation in Gaussian 98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. J. Mol. Struc.: THEOCHEM 1999, 461, 1-21. (46)

Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;

Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (47)

Mafuné, F.; Kohno, J.-y.; Takeda, Y.; Kondow, T.; Sawabe, H. Formation of gold nanoparticles

by laser ablation in aqueous solution of surfactant. J. Phys. Chem. B. 2001, 105, 5114-5120. (48)

Sylvestre, J.-P.; Kabashin, A. V.; Sacher, E.; Meunier, M.; Luong, J. H. T. Stabilization and size

control of gold nanoparticles during laser ablation in aqueous cyclodextrins. J. Am. Chem. Soc. 2004, 126, 7176-7177. (49)

Mafuné, F.; Kohno, J.-y.; Takeda, Y.; Kondow, T.; Sawabe, H. Formation and size control of

silver nanoparticles by laser ablation in aqueous solution. J. Phys. Chem. B. 2000, 104, 9111-9117.



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

(50)

Page 34 of 50

Mafuné, F.; Kohno, J.-y.; Takeda, Y.; Kondow, T.; Sawabe, H. Structure and stability of silver

nanoparticles in aqueous solution produced by laser ablation. J. Phys. Chem. B 2000, 104, 8333-8337. (51)

Scaramuzza, S.; Zerbetto, M.; Amendola, V. Synthesis of gold nanoparticles in liquid

environment by laser ablation with geometrically confined configurations: Insights to improve size control and productivity. J. Phys. Chem.C 2016, 120, 9453-9463. (52)

Amendola, V.; Meneghetti, M. Laser ablation synthesis in solution and size manipulation of

noble metal nanoparticles. Phys. Chem. Chem. Phys. 2009, 11, 3805-3821. (53)

Gehad, G. M.; Nadia, E. E.-G. Preparation and spectroscopic characterisation of metal complexes

of piroxicam. Vib. Spectrosc. 2004, 36, 97-104. (54)

Abd‐Elzaher, M. M. Spectroscopic characterization of some tetradentate Schiff bases and their

complexes with nickel, copper and zinc. J. Chin. Chem. Soc. 2001, 48, 153-158. (55)

Jannesari, Z.; Hadadzadeh, H.; Khayamian, T.; Maleki, B.; Rudbari, H. A. Experimental and

molecular modeling studies on the interaction of the Ru(II)-piroxicam with DNA and BSA. Eur. J. Med. Chem. 2013, 69, 577-590. (56)

Naveenraj, S.; Mangalaraja, R. V.; Wu, J. J.; Asiri, A. M.; Anandan, S. Gold triangular

nanoprisms and nanodecahedra: Synthesis and interaction studies with luminol toward biosensor applications. Langmuir 2016, 32, 11854-11860. (57)

Swierczewska, M.; Lee, S.; Chen, X. The design and application of fluorophore–gold

nanoparticle activatable probes. Phys. Chem. Chem. Phys. 2011, 13, 9929-9941. (58)

Lakowicz, J. R. Radiative decay engineering 5: metal-enhanced fluorescence and plasmon

emission. Anal. Biochem. 2005, 337, 171-194. (59)

Naveenraj, S.; Anandan, S.; Kathiravan, A.; Renganathan, R.; Ashokkumar, M. The interaction of

sonochemically synthesized gold nanoparticles with serum albumins. J. Pharm. Biomed. Anal. 2010, 53, 804-810. (60)

Laban, B.; Zeković, I.; Vasić Anićijević, D.; Marković, M.; Vodnik, V.; Luce, M.; Cricenti, A.;

Dramićanin, M.; Vasić, V. Mechanism of 3,3′-disulfopropyl-5,5′-dichlorothiacyanine anion interaction 33 ACS Paragon Plus Environment

Page 35 of 50

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


with citrate-capped silver nanoparticles: Adsorption and J-aggregation. J. Phys. Chem. C. 2016, 120, 18066-18074. (61)

Laban, B.; Vodnik, V.; Dramićanin, M.; Novaković, M.; Bibić, N.; Sovilj, S. P.; Vasić, V. M.

Mechanism and kinetics of J-aggregation of thiacyanine dye in the presence of silver nanoparticles. J. Phys. Chem. C. 2014, 118, 23393-23401. (62)

Liang, J.; Cheng, Y.; Han, H. Study on the interaction between bovine serum albumin and CdTe

quantum dots with spectroscopic techniques. J. Mol. Struct. 2008, 892, 116-120. (63)

Alanazi, A. M.; Abdelhameed, A. S. A spectroscopic approach to investigate the molecular

interactions between the newly approved irreversible ErbB blocker "afatinib" and bovine serum albumin. PLoS ONE 2016, 11, 1-12. (64)

Beckford, F. A. Reaction of the anticancer organometallic ruthenium compound, η6-p-

cymene)Ru(ATSC)Cl]PF6 with human serum albumin. Int. J. Inorg. Chem. 2010, 2010, 1-7. (65)

Wei, X. L.; Xiao, J. B.; Wang, Y.; Bai, Y. Which model based on fluorescence quenching is

suitable to study the interaction between trans-resveratrol and BSA? Spectrochim. Acta Mol. Biomol. Spectrosc. 2010, 75, 299-304. (66)

Huang, Y.; Zhang, Z.; Zhang, D.; Lv, J. Flow-injection analysis chemiluminescence detection

combined with microdialysis sampling for studying protein binding of drug. Talanta 2001, 53, 835-841. (67)

Zhong, Y.; Deng, C.; He, Y.; Ge, Y.; Song, G. Glutathione-protected silver nanoclusters for

sensing trace-level Hg2+ in a wide pH range. Anal. Methods 2015, 7, 1558-1562. (68)

Gong, A.-Q.; Zhu, X.-S. Determination of epristeride by its quenching effect on the fluorescence

of L-tryptophan. J. Pharm. Biomed. Anal. 2013, 3, 415-420. (69)

Zhang, X. W.; Zhao, F.-L.; Li, K.-A. Studies on the reaction between ciprofloxacin and bovine

serum albumin. Chem. J. Chinese. U. 1999, 20, 1063-1067. (70)

Banerji, B.; Pramanik, S. K.; Pal, U.; Maiti, N. C. Binding of hemoglobin to ultrafine carbon

nanoparticles: a spectroscopic insight into a major health hazard. RSC Adv. 2014, 4, 22536-22541.



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

(71)

Page 36 of 50

El-Sayed, Y.; Gaber, M. Excited state interaction of laser dyes and silver nanoparticles in

different media. Adv. Nanopart. 2012, 1, 54−60. (72)

Sharma, A. S.; Ilanchelian, M. Elucidation of photophysical changes and orientation of acridine

orange dye on the surface of borate capped gold nanoparticles using multi-spectroscopic techniques. Photochem. Photobiol. Sci. 2014, 13, 1741-1752. (73)

Vasimalai, N.; John, S. A. Aggregation and de-aggregation of gold nanoparticles induced by

polyionic drugs: spectrofluorimetric determination of picogram amounts of protamine and heparin drugs in the presence of 1000-fold concentration of major interferences. J. Mater. Chem. B 2013, 1, 5620-5627. (74)

Abdelhalim, M. A. K.; Mady, M. M.; Ghannam, M. M. Physical properties of different gold

nanoparticles: Ultraviolet-visible and fluorescence measurements. J. Nanomed. Nanotechnol. 2012, 3, 16. (75)

Durig, J. R. Vibrational Spectra and Structure; Elsevier, Amsterdam, 1972.

(76)

Liu, W.; Wang, W. D.; Wang, W.; Bai, S.; Dybowski, C. Influence of structure on the

spectroscopic properties of the polymorphs of piroxicam. J. Phys. Chem. B 2010, 114, 16641-16649. (77)

Hamman, J. H.; Demana, P. H.; Olivier, E. I. Targeting receptors, transporters and site of

absorption to improve oral drug delivery. Drug Target Insights 2007, 2, 71-81. (78)

Steichen, S. D.; Caldorera-Moore, M.; Peppas, N. A. A review of current nanoparticle and

targeting moieties for the delivery of cancer therapeutics. Eur. J. Pharm. Sci. 2013, 48, 416-427. (79)

Zhang, Y.; Shareena Dasari, T. P.; Deng, H.; Yu, H. Antimicrobial activity of gold nanoparticles

and ionic gold. J. Environ. Sci. Health C 2015, 33, 286-327. (80)

Shareena Dasari, T.; Zhang, Y.; Yu, H. Antibacterial activity and cytotoxicity of gold(I) and (III)

ions and gold nanoparticles. Biochem. Pharmacol. (Los Angel) 2015, 4, 199. (81)

Panáček, A.; Kvítek, L.; Prucek, R.; Kolář, M.; Večeřová, R.; Pizúrová, N.; Sharma, V. K.;

Nevěčná, T. j.; Zbořil, R. Silver colloid nanoparticles:

Synthesis, characterization, and their

antibacterial activity. J. Phys. Chem. B 2006, 110, 16248-16253.


Page 37 of 50

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


(82)

Wei, D.; Sun, W.; Qian, W.; Ye, Y.; Ma, X. The synthesis of chitosan-based silver nanoparticles

and their antibacterial activity. Carbohydr. Res. 2009, 344, 2375-2382. (83)

El-Baky, R. M. A.; El-Gendy, S. G. Effect of non-steroidal anti-inflammatory drugs and

dexamethazone on the biofilm formation and expression of some adhesion-related genes of Candida albicans and Staphylococcus aureus. Afr. J. Microbiol. Res. 2016, 10, 694-707.



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

Page 38 of 50

Table 1. IC50 values (μg/mL) for the compounds towards the Jurkat T-cells. Compound

IC50

Au NPs

> 200

Pir

> 200

Au NPs–Pir

33.80 ± 2


Page 39 of 50

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


Figure Captions Figure 1. TEM image of the gold nanoparticles (Au NPs) prepared in Pir aqueous solution (10–4 M). Figure 2. Absorbance spectra of the free Pir and Au NPs–Pir samples in different intervals Au NPs generation. Figure 3. Fluorescence spectra of the Pir solution (10–4 M, λex = 260 nm) in the presence of different concentrations of the Au NPs (0.0 to 1.1 × 10−8 M). Figure 4. Emission spectra of the Au NPs (λex = 510 nm and λem = 765 nm) in different intervals Au NPs generation for the in situ method. Figure 5. FT–IR spectra of the free Pir (red line) and the adsorbed Pir (Au NPs–Pir) (black line). Figure 6. The optimized structures along with the numbering of atoms of different Au atom–Pir conjugates (In these structures (I-V), an Au atom is interacting with the different probable binding sites of Pir). Figure 7. The calculated IR spectrum of the free Pir along with the IR spectra of the structures shown in Figure 6. Figure 8. The optimized structure of Pir on the Au(111) surface. Figure 9. The juxtaposition of the optimized structure of the free Pir on the deformed structure of Pir (D–Pir) after the adsorption on the Au surface. Figure 10. The cytotoxic effect of the Au NPs, Pir, and Au NPs–Pir against the Jurkat T-cells in a concentration-dependent manner.



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

Page 40 of 50

Figure 1. TEM image of the gold nanoparticles (Au NPs) prepared in Pir aqueous solution (10–4 M).


Page 41 of 50

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


Figure 2. Absorbance spectra of the free Pir and Au NPs–Pir samples in different intervals Au NPs generation.



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

Figure 3. Fluorescence spectra of the Pir solution (10–4 M, λex = 260 nm) in the presence of different concentrations of the Au NPs (0.0 to 1.1 × 10−8 M).


Page 42 of 50

Page 43 of 50

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


Figure 4. Emission spectra of the Au NPs (λex = 510 nm and λem = 765 nm) in different intervals Au NPs generation for the in situ method.



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

C13=O14 C=C;C=N (Pyridyl ring) C23=O27

C5–N11

C23–C26

O=S=O asymmetric

Figure 5. FT–IR spectra of the free Pir (red line) and the adsorbed Pir (Au NPs–Pir) (black line).


Page 44 of 50

Page 45 of 50

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


I

II

III

IV

V

Figure 6. The optimized structures along with the numbering of atoms of different Au atom–Pir conjugates (In these structures (I-V), an Au atom is interacting with different probable binding sites of Pir).



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

Page 46 of 50

Free Pir O=S=O C5–N11 C23–C26 C23=O27 Pyridyl ring

C13=O14

I

C13=O14

O=S=O C5–N11 C23–C26 Pyridyl ring

C23=O27

II

O=S=O Pyridyl C5–N11 ring C23=O27 C23–C26

C13=O14

III O=S=O Pyridyl ring C5–N11 C23–C26

C13=O14

C23=O27

IV Pyridyl ring C13=O14

O=S=O C5–N11 C23–C26

C23=O27

V O=S=O C5–N11 C23–C26 Pyridyl C13=O14 ring C23=O27

Figure 7. The calculated IR spectrum of the free Pir along with the IR spectra of the structures shown in Figure 6. 45 ACS Paragon Plus Environment

Page 47 of 50

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


2.37Å

2.43Å

2.49Å

Figure 8. The optimized structure of Pir on the Au(111) surface.



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

Page 48 of 50

D–Pir Free Pir

Figure 9. The juxtaposition of the optimized structure of the free Pir on the deformed structure of Pir (D–Pir) after the adsorption on the Au surface.


Page 49 of 50

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


Figure 10. The cytotoxic effect of the Au NPs, Pir, and Au NPs–Pir against the Jurkat T-cells in a concentration-dependent manner.



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

“TOC Graphic”


Page 50 of 50

Combined Experimental and Computational Study of the In Situ

Recommend Documents