Colloidal PEGylated Bimetallic Au-Ag Nanoparticles | International News Network

2021-11-12 10:53:06 By : Ms. Sunny Cao

Javascript is currently disabled in your browser. When javascript is disabled, some functions of this website will not work.

Open access for scientific and medical research

From submission to the first editing decision.

From editor acceptance to publication.

The above percentage of manuscripts have been rejected in the past 12 months.

Open access to peer-reviewed scientific and medical journals.

Dove Medical Press is a member of OAI.

Batch reprints for the pharmaceutical industry.

We provide real benefits for authors, including fast processing of papers.

Register your specific details and specific drugs of interest, and we will match the information you provide with articles in our extensive database and send you a PDF copy via email in a timely manner.

Back to Journal »International Journal of Nanomedicine» Volume 16

One-pot synthesis of PEGylated bimetallic gold and silver nanoparticles for oral cancer imaging and radiosensitization

Author Ahmed S, Baijal G, Somashekar R, Iyer S, Nayak V

Published on October 21, 2021, the 2021 volume: 16 pages 7103-7121


Single anonymous peer review

Editor approved for publication: Dr. Phong A Tran

Shameer Ahmed, 1 Gunjan Baijal, 2 Rudrappa Somashekar, 3 Subramania Iyer, 4 Vijayashree Nayak 1 1 Department of Biological Sciences, Birla College of Technology and Science, Pilani, KK Birla Goa Campus, Sancoale, Goa, India; 2 Manipal, Goa, India Department of Radiation Oncology, Hospital Goa; 3 Material Science and Technology Center, Vijnana Bhavan, Mysore, Karnataka, India; 4 Head and Neck Oncology Department, Poekkara Amrita Institute of Medical Sciences, Cochin, India Corresponding author: Vijayashree Nayak Department of Biological Sciences, Birla Technology and Science College Pilani, KK Birla Goa Campus, NH 17B, Bypass Road, Zuarinagar, Sancoale, Goa, 403726, India Tel 91 832 258 0101 (Ext. 161) Email [email protected] Background: Radiotherapy is the treatment of a variety of head and neck squamous forms An important way of cell cancer. Nanomaterials composed of high atomic number (Z) elements are a new type of radiosensitizer that can enhance radiation damage by generating free radicals and subsequent DNA damage. Gold nanoparticles are promising radiosensitizers due to their high (Z) biocompatibility and easy surface engineering. Compared with single metal nanoparticles, bimetal nanoparticles show enhanced anticancer activity. Materials and methods: A simple one-pot synthesis technique was used to synthesize PEG-coated Au-Ag alloy nanoparticles (BNPs). The size measured by dynamic light scattering is approximately 50 ± 5 nm. Analyze morphology, structure composition and element mapping by electron microscope and SAXS (Small Angle X-ray Scattering). The radiosensitization effect on KB oral cancer cells was evaluated by 6MV X-ray irradiation on a linear accelerator. Stain cells using a confocal microscope with Hoechst stain to image nuclear damage. Compare BNP's computed tomography (CT) contrast enhancement with the clinically used drug Omnipaque. Result: BNP was synthesized using PEG 600 as reducing agent and stabilizer. The surface charge of a well-dispersed colloidal BNPs solution is -5mV. The electron microscope showed a spherical shape. HAADF-STEM and element mapping studies show that the constituent metal is a mixed nano-alloy of Au and Ag. Due to the absorption of the PEG layer and water molecules, the hydrodynamic diameter is about 50±5nm. SAXS measurement confirmed that the size of BNP is about 35nm. When BNP was coated with PEG, a Raman shift of approximately 20 cm-1 was observed. 1H NMR shows that -OH is widely involved in the synthesis. BNPs effectively enter the cytoplasm of KB cells and show effective external radiosensitization with an enhancement rate of about 1.5-1.7. Imaging Hoechst stained nuclei showed apoptosis in a dose-dependent manner. Compared with Omnipaque, BNP shows better CT contrast enhancement capabilities. Conclusion: This kind of bimetallic hybrid nanoparticles can play a dual function as a radiosensitizer and CT contrast agent to fight oral cancer, and may also be used for other cancers. Keywords: bimetallic mixed alloy nanoparticles, radiosensitizer, nanomedicine, PEG, polyethylene glycol, CT contrast, cell apoptosis

Oral cancer refers to tumors diagnosed in the lips, the anterior 2/3 area of ​​the tongue, the upper and lower alveolar ridges, the hard palate, the buccal mucosa, the posterior triangle area of ​​the molars, and the sublingual area (including the floor of the mouth). Among cancers, approximately 80-90% are histopathologically differentiated mucosal epithelial squamous cell carcinoma, also known as oral squamous cell carcinoma (OSCC). 2 Radiotherapy (RT) is chosen as a radical treatment, or in some cases as the main way to treat primary oral cancer after surgery. The decrease in saliva production can have a negative effect. Salivary glands can be protected by barrier methods, such as IMRT (Intensity Modulated Radiotherapy) shielding or retention techniques, which reduce the incidence and a certain degree of xerostomia. 3 Radiosensitization is to improve the radiation damage to cancer cells while limiting the radiation effect on adjacent normal tissues; the use of radiosensitizing compounds to develop tumor cell cycle, metabolism and microenvironmental characteristics can selectively enhance the damage to cancer cells . 4 Heavy metal nanoparticles with high atomic number (Z) values ​​are promising radiosensitizers because they can scatter, absorb, and emit radiant energy. The synthesis of these is a robust process through which the required size and shape can be achieved. They also have the following advantages, such as low cytotoxicity, good biocompatibility and surface functionalization through bioengineering. 5 Gold nanomaterials have shown good effects in various tumors, meeting the standards. 6 Other high-Z nanomaterials have also been tested as radiosensitizers, such as silver and hafnium oxide nanoparticles. 7-9 The size of nanoparticles plays a crucial role, such as increasing penetration and retention behavior. The performance of 50nm metal nanoparticles of gold, silver and hafnium oxide nanoparticles is better than other sizes in terms of enhancing the radiosensitization effect on cancer cell formation. 10-13 Bimetallic colloids arouse the interest of researchers due to their interesting structure and unique catalytic, electronic and optical properties. 14 Bimetallic nanoparticles based on structure are divided into: (i) core-shell alloys, (ii) clusters in clusters, and (iii) random alloys and alloy structures. The size and shape of bimetallic nanoparticles depend particularly on their synthesis technology and manufacturing conditions. 15 Radiosensitization plays an important role in the treatment of most cancers and is regarded as the "new dogma of cancer treatment." 72 In preclinical studies, gold nanoparticles (AuNPs) have proven to be promising drug candidates as radiosensitizers. 16 A study conducted on the KB cell line used various shapes of thiol-PEG functionalized gold nanoparticles while maintaining the size around 50nm. It was found that spherical Au NPs were better at internalizing cancer cells than spike-shaped and rod-shaped gold nanoparticles particle. In addition, the SER (sensitization enhancement rate) of spherical Au NPs is about 1.62 higher among the three NPs. 74 Recent studies have shown that bimetallic NPs act as a better radiosensitizer than the corresponding single metal NPs; Compared with cells exposed to radiation (2-10 Gy, 200 kVp), when exposed to ZnO/SiO2 NPs, the radiation-induced cell killing was enhanced by a factor of two. 17 Au in combination with titanium shows ~1.8 and ~1.2 when the particles are fabricated on carriers with sizes of 10-30 nm and 5-10 nm. The mechanism of 18 AuNPs radiation sensitization is that it enhances the radiation effect through biological and physical interaction with IR (ionizing radiation). In the physical phase of interaction, AuNPs cause damage to cells by increasing the production of photoelectrons, Auger electrons, and low-energy secondary electrons. In the chemical stage, the surface of the electronically active AuNPs will lead to catalysis and the formation of free radicals including reactive oxygen species (ROS), and extremely low-energy electrons make nuclear components and DNA chemically sensitive to damage caused by infrared rays. Finally, in the biological stage, gold nanoparticles enhance the effect of IR through DNA repair inhibition, cell cycle interruption and oxidative stress. 19 Cancer radiosensitization is an event that relies on the internalization of nanoparticles in cancer cells. This process is directly affected by the following factors: 20 Previous studies have shown that gold-silver bimetallic nanoparticle alloys also have enhanced anticancer activity. The mechanism and pathway of cytotoxicity is to activate caspase and p53/Bax/Bcl-2 apoptotic pathways on various cancer cell lines. 21-24 PEG (poly[ethylene glycol]) is cost-effective and has been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) due to its excellent water solubility, low toxicity, and environmentally friendly polymer. 25 Used in various applications. Recently, since it interacts with metal NPs through electrostatic and spatial effects, it has been used to make metal NPs. 26,27 NPs whose surface is modified or functionalized with PEG can prolong blood circulation time and reach target sites. 28 Surface functionalized NP can improve the quality of radiosensitizers and counteract the molecular blockade of cell death. Histidine-anchored gold nanoclusters (Au NCs-His) were shown to be radiosensitizers with a high sensitization rate of about 1.54. The mechanism is that Au NCs-His can reduce the level of intracellular GSH and enhance the production of intracellular reactive oxygen species (ROS). 75 Another study using in vitro and in vivo models showed that gold nanospikes (GNS) functionalized with thiol PEG and peptide molecules TAT ​​have a very high SER, about 2.30; the underlying mechanism of this SER is to increase (ROS), mitochondrial depolarization and cell cycle redistribution. 76 In addition, the efficiency and efficacy of metal-based nanoformulations depend on the source and type, such as X-rays or gamma rays, and the radiation energy, amount, and location of nanomaterials within the tissue. Active targeting through the coating and positioning of ligands or antibodies and the pharmacokinetic characteristics of NPs will produce more effective treatments with less adverse effects on surrounding healthy tissues. 29,30 In recent years, gold-silver nanoalloys or gold-silver bimetallic nanoparticles have obtained special characteristics due to their unique characteristics compared with single-metal systems, such as anticancer agents, radiosensitizers and contrast agents for imaging , So attracted attention. 31-35

In order to explore the advantages and synergistic properties of the alloy, this is the first of its kind. We report here the colloidal PEGylated bimetallic Au-Ag nanoparticles using PEG 600 to reduce HAuCl4 and AgNO3 simultaneously through the polyol method. One-pot synthesis. These nanoparticles were thoroughly characterized to establish structures and nanostructures, and showed anticancer activity, nuclear damage, and potential radiosensitizers for oral cancer cell lines. The phantom model demonstrates the presence of CT nanocontrast agents because of the presence of gold compared to iodine-based fully iodinated CT contrast agents.

Gold(III) chloride trihydrate (HAuCl4·3H2O, 99.9%) and silver nitrate (AgNO3, 99.9%) as precursors, polyethylene glycol {PEG} (H2O-[CH2-CH2-O]nH), With a molecular weight of 600 (bio ultra, PEG 600), it is used as a reducing agent and stabilizer. Sodium hydroxide particles were purchased from Fisher Scientific and dissolved in water and used to change the pH. Use Milli Q water (purity up to 18.2 MΩcm-1 Elga Option Q7). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), antibiotic antifungal solution, gentamicin solution and trypsin-EDTA solution were purchased from Himedia Laboratories (Mumbai, India). Used in the following studies: 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bis-1H-benzimidazole trihydrochloride ( Hoechst 33258) hydrate, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and deuterium. The 77 KB cell line was purchased from the National Center for Cell Science (NCCS), Pune, India. The chemicals used in the experiment are molecular grade and analytical grade. Unless otherwise stated, all reagents were used without further purification.

BNP with an average hydrodynamic size of 50±5nm was synthesized using PEG 600. Add 1% sodium hydroxide NaOH (350 µL) to the mixture of PEG 600 (500 µL) and water (50 mL) in an Erlenmeyer flask. Add a mixture of AgNO3 (1mM, 3.5mL) and HAuCl4 (1mM, 3.5mL) to it, and boil it on a hot plate at approximately 140°–180°C for 10 minutes. Turn off the heating plate and leave the flask on the heating plate for another 5 minutes as a holding time for gradual cooling. The color of the flask changed to orange, indicating the formation of PEG-reduced and stable bimetallic gold-silver nanoparticles. Measure the pH value and synthesize in triplicate. Excess PEG and unreacted reagents were removed from the colloidal solution by centrifuging the pellet at 12,000 rpm for 30 minutes, removing the supernatant, and resuspending the BNP in Milli Q. Washing is performed twice. BNP is a stable colloid for more than 1.5 years after synthesis. The measured pH is approximately 7.2.

Dynamic light scattering (DLS) is used to measure the hydrodynamic size of BNP. Microtrac Nanotrac Wave (USA) was used for particle size and zeta potential analysis. The hydrodynamic diameter of colloidal particles and surface conjugates can be evaluated by DLS. Transmission Electron Microscope (TEM), High Resolution, Transmission Electron Microscope (HR-TEM), Selected Area Electron Diffraction (SAED), High Angle Annular Dark Field (HAADF), and element mapping, that is, shape, form, size and use at 200 kV A transmission electron microscope (Titan Themis 300kV from FEI, now Thermo) operated under the microscope studied the diffraction patterns of PEG-reduced and capped nanoparticles. Laboratory-based small-angle X-ray scattering (SAXS) facilities are used to study nanoparticles. The concentration of BNP was determined by using inductively coupled plasma mass spectrometry (ICP-MS) Agilent ICP-MS 7900 and (UHMI) ultra-high matrix introduction. The UV-Vis spectrum of the synthesized BNP was recorded on a UV-Vis spectrometer (Multiskan™ GO Microplate Spectrophotometer, Thermo Fisher Scientific), using a standard orifice plate at room temperature, with a spectral range of 200nm to 900nm and a spectral resolution of 2nm. The Raman spectroscopy experiment was performed on the XPlora PLUS spectrometer (Horiba Scientific, France). The Raman spectrum was recorded at room temperature using an excitation wavelength of 532nm (diode laser). The resolution of the spectrum is 2 cm-1. All NMR spectra were recorded by Agilent 400-MR DDR2 (Varian, Palo Alto, USA) spectrometer, and the solvent used was DMSO for calibration. The chemical shift is in ppm.

In terms of cell culture, KB cells (derived from oral epidermal cancer) contain 5% CO2 and maintain an exponential growth state. The cells were passaged using 0.25% trypsin. Approximately 1×104 KB cells/well are cultured in 96-well plates. After 24 hours, remove the medium and add fresh medium containing 0 to 0.34μg/mL BNP to the test wells, and place the cells in the incubator for another 24 hours. Each concentration was tested in triplicate. In addition, the experiment was performed at three different time points. Cells without nanoparticles were used as a control. At the end of the incubation period, remove the medium and perform MTT determination: add 50 μL of MTT solution (5 mg/mL) to each well and incubate for 3 hours. Then, remove the excess MTT and add 100 µL DMSO to dissolve the formazan crystals. Measure the absorbance at 570nm in the Multiskan™ GO microplate reader (Thermo science, USA); all cytotoxicity methods and procedures have been completed. 77

Culture approximately 1×104 KB cells/well in various 96-well plates. After 24 hours, remove the medium and add fresh medium, then label the 96-well plate. Using a Versa HD® (Elekta, Crawley, England) linear accelerator equipped with an Agility® collimator system to irradiate cells with 6 MV photon X-rays, as a supporting information Figure S3 shows a photo of the LINAC equipment used in this study, located in Manipal Hospital , Dona Paula, Goa. The source-to-surface distance (SSD) is 100 cm, the field of view is 20×20 cm2, and the total dose of 2, 4, 6 and 8 Gy is delivered to various 96-well plates at a dose rate of 200 MU min-1. After irradiation, MTT measurement was performed, and cell viability was calculated. To test the radiosensitization ability of BNPs, KB cells were cultured in 96-well plates at a density of 1×104 cells/well for 24 hours. After 24 hours, remove the medium and add fresh medium containing 0 to 0.34μg/mL BNP to the test wells, and place the cells in the incubator for another 24 hours. After that, the medium was replaced with fresh medium, and the BNP of the non-internalized KB cells was washed away. KB cells with internalized BNP were irradiated with 4Gy X-rays and then incubated for 24 hours. Finally, use the MTT assay to check cell viability. Each concentration was tested in triplicate. In addition, the experiment was conducted at three different time points.

The use of 4, 6-diamidino-2-phenylindole (DAPI) staining and fluorescence microscopy can well record radiation-induced nuclear damage, such as apoptosis, mitotic catastrophe and cell senescence, and the following experiments are almost unchanged . 36 In short, KB cells and coverslips are seeded in a 6-well plate with 3×105 cells per well, incubated at 37°C and 5% CO2 for 24 hours, and then fresh medium and 0.34μg/well are added. mL BNP, use H2O2 as a positive control, and only the medium as a control, incubate at 37°C and 5% CO2 for 24 hours. Replace all cells with fresh medium, irradiate the cells with 4Gy X-rays and incubate for 24 hours. After incubation, fix the cells with 3% paraformaldehyde 2% sucrose fixative solution and wash twice with PBS (1X). Then the cells on the coverslip were counterstained with Hoechst 33258 diluted 1:5000 in PBS. The stained cover glass was poured on a glass slide, then fixed with an adhesive, and then observed under a fluorescence microscope (Olympus Corporation FV3000).

The stability of BNP is evaluated by calculating the change in turbidity in FBS. Add 150 μL of BNPs suspension (0.34 μg/mL) to 150 μL of FBS in a 96-well plate, and incubate in a 37 °C incubator for different time points, up to 72 hours. Thereafter, the absorbance of each sample was measured at 415nm. A 5% glucose solution was used as a negative control. 37

For CT imaging and HU measurement, a 1.5 mL centrifuge tube was used to make a phantom model, and 0.6 mg/mL BNP was filled in Milli Q, 1x PBS, and Milli Q as a control. The concentration of Novaplus Omnipaque and CT contrast medium 2 used in clinical practice was 5 mg/mL And 10mg/mL. A Philips Ingenuity (PI) 64-slice MDCT scanner (Philips Medical Systems, Massachusetts, USA) was used to obtain CT data. In the supporting information, Figure S4 shows a photo of the CT instrument used to image the phantom. The imaging parameters are as follows: effective pixel size = 1.0mm; 120 kVp, 399 mA; field of view (FOV) = 170 mm × 190 mm; number of rotation steps = 180; exposure time = 150 milliseconds/rotation. Weasis v1.2.8 software was used to analyze the CT analysis images of the phantom. The calculation of the value is done by recording Hounsfield Units (HU) from a randomly selected region of interest.

The data about cell viability was entered into Microsoft Excel and analyzed using IBM SPSS (Statistical Package for Social Sciences) Windows Statistical Data Version 20 (IBM Corp., Armonk, NY, USA). The Kolmogorov-Smirnov test was used to test the normality of the analyzed data. Determined descriptive statistics. Cell viability was measured when using different concentrations of BNP, BNP 4Gy and X-ray doses. The percentages of apoptosis and mitotic catastrophe were analyzed in BNP, 4Gy, BNP 4Gy, H2O2, H2O2 4Gy and the control group. Using Kruskal-Wallis H statistical test followed by Dunn-Bonferroni post-hoc test (α = 0.05) for multiple comparisons, different concentrations of cell death survival rate (%), apoptosis percentage and mitotic disaster were analyzed. The statistical significance level was determined as p<0.05. All measurements were performed in triplicate and expressed as mean ± standard deviation (SD).

The synthesis of PEGylated bimetallic Au-Ag NPs is shown in Figure 1. The process of synthesizing NPs is kept simple and uses the one-pot method. This is a typical polyol reaction in which the metal precursor is reduced and stabilized by polyethylene glycol. The synthesis of BNPs (Au-Ag) is a chemical reaction in which silver and gold precursors are simultaneously reduced at elevated temperatures (160°–180°C), and 1% NaOH is used to adjust the pH. PEG-coated or encapsulated BNP has steric repulsion, which can prevent them from agglomerating. The presence of -OH groups changes the pH value to an alkaline environment, and promotes the rapid reduction of Au3 and Ag ions to Au0 and Ag0, respectively; pH ​​changes can change the size parameters, such as the size and shape of the nanoparticles being formed. 38 A solution with a higher pH value will accelerate the decomposition of AuCl3, thereby accelerating the reduction of gold ions by PEG. The mechanism for this is due to the oxidation of hydroxyl end groups to aldehyde groups, forming PEGylated Ag NPs in the silver precursor. Without NaOH, the reaction cannot proceed forward. 39,73 A small amount of Au3 was released from the polygonal Au particles during 10 minutes of heating from the ambient temperature to a high temperature of 150°C. At the same time, maintaining this temperature for 10 minutes will cause the surface of the Au particles to dissolve in the presence of H and NO3- ions. In the presence of PEG, Ag ions are reduced and form H ions. (1) (2) Figure 1. Schematic diagram of the synthesis of bimetallic Au-Ag nanoparticles in PEG 600 solution. Metal ions combine with reducing metabolites/stabilizers and are reduced to metal atoms at the same time. At the optimal pH, metal ions and metabolites interact with similar complexes to form a small bimetallic nanoparticle. Next, during nucleation, they grow and coalesce to form mixed clusters of gold and silver. The reaction proceeds until the particles reach a stable size and shape.

Figure 1 Schematic diagram of the synthesis of bimetallic Au-Ag nanoparticles in PEG 600 solution. Metal ions combine with reducing metabolites/stabilizers and are reduced to metal atoms at the same time. At the optimal pH, metal ions and metabolites interact with similar complexes to form a small bimetallic nanoparticle. Next, during nucleation, they grow and coalesce to form mixed clusters of gold and silver. The reaction proceeds until the particles reach a stable size and shape.

In addition, NO3- ions are formed by the electrolytic dissociation of AgNO3. Au3 and Ag ions are simultaneously reduced to form uniform Au-Ag nano-alloy hybrid nanoparticles. The results show that some spherical Au particles or exccentre [email protection] particles are attached to the surface of silver-rich Ag/Au alloy nanowires to reduce the total surface energy (Osterwald ripening). Therefore, they are easier to fuse. Other possible reasons may be that a certain amount of Au particles are dissolved by etching, and Au3 ions are generated from the surface of the Au particles. The standard cell potential of the following reactions is positive. Finally, if the Au3 cation is released, the dissolution reaction of Ag(s) will spontaneously proceed in the nearby part. (3)

When Au-Ag/Au particles grow to a critical size (~400nm), they are stable to thermal annealing, and monodisperse Au-Ag/Au particles are the final product. 40

The ultraviolet-visible absorption spectra of BNPs indicate that the surface plasmon resonance (SPR) band is located between the fingerprint regions of Au and Ag nanoparticles. The optical properties of BNPs exhibit plasmon resonance at 479nm, which indicates that nanoparticles are likely to be the characteristics of Au-Ag mixed alloys in nature. Figure 2A shows the absorption spectrum of colloidal BNP. The maximum peak of BNPs indicates a homogeneous colloidal mixing of gold and silver nanoparticles without the formation of individual gold or silver particles. Figure 2B is a digital photo of the brown-orange colloidal BNP. 35 In the supporting information, Figure S1, we use UV-Vis spectroscopy to show the reproducibility of synthesized BNP at three different time points. PEG is a stabilizer, which effectively prevents the agglomeration of newly formed nanoparticles. The size of synthetic nanoparticles can be determined by SPR bands. The frequency and width of SPR depend on the shape and size of the nanoparticles, the surrounding medium, and the dielectric constant of the metal. 41 Figure 2 (A) UV-Vis spectra of PEGylated bimetallic Au-Ag NPs (BNPs) and (B)) Picture of synthesized BNP (orange-brown appearance).

Figure 2 (A) UV-Vis spectra of PEGylated bimetallic Au-Ag NPs (BNPs) and (B) a picture of the synthesized BNPs (the appearance is orange-brown).

Figure 3A shows the particle size distribution (PSD) of BNP measured using dynamic light scattering (DLS). The average hydrodynamic diameter of BNP is 50±5nm. BNPs are retained in Milli Q as monodisperse colloids with a hydrodynamic diameter of 50nm. ImageJ software was also used to measure the size of BNP from TEM images, and the measurement results were comparable to those obtained from DLS. The difference between Dhydro and DTEM (the size of the dried particles obtained by TEM) is due to the binding and capping of PEG 600 to the surface of the nanoparticles; Dhydro-DTEM is corona, about 15nm. As shown in Figure 3B, the electrophoretic mobility measurement value of BNP is recorded by the zeta potential value obtained by Zetasizer. The zeta potential range of particles in Milli Q water is -5.6 mV. Since PEG is hydrophilic in nature, the low negative surface charge may be caused by the presence of absorbed water molecules. The negative zeta potential indicates the negatively charged surface ligand on the colloidal BNP. The stability of dispersed nanoparticles depends on their pH and surface charge. 42 The PEG chains on the particle surface are stable due to strong spatial repulsion, which is opposite to electrostatic repulsion, which is usually insensitive to different ions nearby. 43 Figure 3 (A) Hydrodynamics of 50±5 nm BNP in Milli Q water Diameter (Dhydro) (Manually measure pH~7.4) (B) Zeta potential -5.6mV and mobility.

Figure 3 (A) Hydrodynamic diameter (Dhydro) of 50±5 nm BNP in Milli Q water (pH~7.4 manual measurement) (B) Zeta potential -5.6mV and mobility.

Figure 4A and B show the TEM image and particle size distribution histogram of BNP, respectively. All nanoparticles are nearly spherical and well dispersed. The size distribution analysis based on the TEM image produces an average value of approximately 36 ± 5 nm. Due to the contrast difference caused by the atomic number Z of silver and gold (Ag is 47; Au is 79), TEM confirmed the bimetallic structure of BNP between silver and gold. 44 Figure 4 (A) TEM image of BNP and gold (B) The histogram shows the particle size distribution of BNP.

Figure 4 (A) TEM image of BNP and (B) histogram showing the particle size distribution of BNP.

The scattered intensity is recorded as a function of the scattered wave vector transfer Q (= 4π sinθ/λ, where 2θ is the scattering angle and λ is the X-ray wavelength). The Q range of the instrument is 0.1–2.3 nm-1. In the case of X-rays, as shown in Figure 5, compared to polymers (low Z component), the scattering mainly comes from the bimetallic part (high Z component). The data is fitted with a BNP diameter equal to 35.2 nm. The supporting information shows the SANS operating parameters and BNP's SANS analysis (including calculations and spectrum) (Figure S2 provides an in-depth understanding of the experiment). Figure 5 SAXS data of PEGylated bimetallic Au-Ag NPs (BNPs). The solid line is the theoretical fit to the experimental data.

Figure 5 SAXS data of PEGylated bimetallic Au-Ag NPs (BNPs). The solid line is the theoretical fit to the experimental data.

Figure 6A shows the HRTEM image of PEGylated bimetallic Au-Ag NPs. The obvious difference in contrast between the brighter and darker areas means the formation of bimetallic Au-Ag NP. In addition, multiple lattice fringes with a lattice spacing of 0.20nm are indexed to the (200) plane of the face-centered cubic (FCC) of silver, and the measured lattice spacing of 0.23nm is in good agreement with the (111) plane of the FCC Match gold. 45,46 Powder XRD is a powerful tool for characterizing the crystal structure of nanomaterials. According to the position of the 2θ value of the corresponding metal, it is easy to figure out the structure (alloy, core/shell or physical mixture) and crystallinity of the prepared nanoparticles. Since the lattice constants of Ag and Au are very similar (4.086 Å for Ag; 4.078 Å for Au), this method is particularly difficult to apply to the structure and characterization of existing BNPs. In addition, the presence of PEG polymer shows an amorphous peak. Therefore, the SAED pattern is recorded and shown in Figure 6B. The SAED pattern shows that BNP is a nanocrystal grown along (111), (200), (220) and (311) crystal plane size, with corresponding 2θ.47 Figure 6 (A) PEGylated bimetallic Au-Ag NPs HRTEM image and lattice spacing of Au-Ag NPs nanocrystals. (B) SAED mode of PEGylated bimetallic Au-Ag NP Figure 7 (A) HAADF-STEM image of BNP inserted into a set of EDX spectra of BNP. The (CF) HAADF image shows the mosaic of Au-Ag EDS element mapping, showing the mixture of two elements in the nanoparticle group.

Figure 6 (A) HRTEM image of PEGylated bimetallic Au-Ag NPs and the lattice spacing of Au-Ag NPs nanocrystals. (B) SAED mode of PEGylated bimetallic Au-Ag NP

Figure 7 (A) Insert (B) HAADF-STEM image of BNP in EDX spectrum from a set of BNP. The (CF) HAADF image shows the mosaic of Au-Ag EDS element mapping, showing the mixture of two elements in the nanoparticle group.

In Figure 7C, the HAADF image of BNP shows the contrast between Au and Ag elements; the contrast is due to Rutherford scattering. The signal in the HAADF-STEM mode depends on the atomic number (Z) of the element, low brightness refers to Ag (lighter element), and strong brightness corresponds to Au (heavy element). 48 The image clearly shows the Ag and Au elements in the mixed distribution of BNP. The EDS element mapping analysis in HAADF-STEM in Figure 7D and E confirms their structure: the chemical composition of Ag and Au in the presence of Au signal (red) and Ag signal (blue) are shown in the bimetallic nanoparticles. The HAADF image and EDX mapping in Figure 7F show the coexistence of nano-alloyed silver and gold within the nanoparticles in BNP. In addition, the EDX spectrum and element mapping of the particle pool are regarded as important examples of BNP. It explains the alloy type of the synthesized Au-Ag particles. EDX confirmed the distribution of two metals in BNP, and no single metal particles were seen. The EDS spectrum of the bimetallic Au-Ag NPs is shown in Figure 7B. The HAADF-STEM of the BNPs in Figure 7A has an insert. The peaks shown in the BNP spectrum correspond to Ag 3.00 eV and 3.15 eV, while the peaks at 2.0, 9.5 and 11.5 keV correspond to Au.49

In addition, inductively coupled plasma mass spectrometry (ICPMS) is used to quantify the gold and silver content in BNP. As shown in the supporting information (Table T1-ICPMS Calibration and T2-Working Parameters), the BNP (in ppb unit) 131 and 205 solutions of silver and gold content in the standard solutions were measured using ICP-MS, respectively (ICPMS).

Figure 8A shows the vibration mode characteristics of the PEG 600 peaks at 471, 557, 883, 1013, 1240, 1283, 1479, 2563, 2700, 2878, 2940, and 3458 cm-1. In addition, Figure 8B shows the Raman spectrum of PEG 600 in the 400–600 cm-1 region. The Raman peak and the corresponding peak in the Raman spectrum of PEG alone have the most phase shift (15–20 cm-1); such regions shown here are 480 to 500 cm-1 and 502-520 cm-1, indicating that the figure The PEGylation of BNP is shown in 8C. This phenomenon is common in SERS; if ligands, molecules, or drugs are chemically adsorbed to the surface of nanoparticles, they will experience an increase and change in signal at reduced concentrations. In a similar study, the drug excipients of the Raman spectrum of the colloidal vibration mode of AgNPs with SDS characteristics showed a Raman shift of 20 cm-1. 50 BNP PEG on the metal surface may increase the oscillator strength and orientation of the molecule. 51 This proves that controlled aggregation can significantly increase the SERS response of silver nanoparticles. The dimer nanoparticles of BNP and the presence of silver play a role in the formation of hot spots. In addition, the addition of a low zeta potential of approximately -5.6mV may result in the formation of a dimer configuration during the characterization process, as shown in the TEM image. The high-performance SERS of 52 nanoparticles indicates the Au-Ag bimetallic superstructure. The combined effect (dimer formation shown on the UV-vis spectrum) between the two noble metals and the abundant “hot spots” shown in Figure 8D creates a superstructure that enhances the SERS detection of the Ag shell and makes Au -Ag superstructure is very attractive. 53 Figure 8 (A and B) Raman spectra collected from PEG 600 and (C) BNP and (D) UV-Vis spectra, depicting the formation of hot spot dimers.

Figure 8 (A and B) Raman spectra collected from PEG 600 and (C) BNP and (D) UV-Vis spectra, depicting the formation of hot spot dimers.

The spectrum in Figure 9A confirms the structure of PEG 600; 1H NMR (400 MHz, DMSO) δ 4.55 ppm is the hydroxyl triplet, and δ (3.30–3.47 ppm) is attributed to the methine proton signal of PEG and is regarded as PEG The spectrum of synthesized BNPs with main chain 25 and DMSO d6 δ 2.55 ppm. 54,55 Figure 9B shows the DMSO d6 δ 2.55 ppm and 3.35–3.45 ppm methine protons of the polyethylene glycol unit, but the characteristic signal hydroxyl peak δ 4.55 ppm Has disappeared. The possible explanation is that -OH is involved in the synthesis and is encapsulated in the corona of PEG capsules or nanoparticles. It is also obvious that the zeta potential in BNP is smaller because the -OH depletion of the hydroxyl end group has already occurred, resulting in little capture on the surface and the rather weak normal CH3 methyl group surrounding the NPs and fewer -OH groups, because Less negative zeta potential of -5.6mV. 56 Figure 9 1H NMR (A) PEG 600 (B) PEG-coated BNP in DMSO-d6 at 400 MHz.

Figure 9 1H NMR (A) PEG 600 (B) PEG-coated BNP in DMSO-d6, the frequency is 400 MHz.

KB cells were treated with six low concentrations of 50nm BNP for 24 hours without X-ray treatment, and MTT assay was used to determine cell viability. Except that 0.34 and 0.17 µg/mL BNP showed slight toxicity, there was no significant difference between the total cell number of control cells (Figure 10A). The cytotoxic potential of NPs depends on the materials or elements used or arranged. The shape of the nanoparticle plays a vital role in determining the toxicity of the nanoparticle. The toxic effect of silver depends on the shape, and nanotriangles show more damage than nanospherical silver NPs. Nanomaterials have a larger surface area, leading to higher toxicity and reaction potential. The uptake of NP by cells depends on factors such as size, shape, zeta potential, nature of capping agent, carrier and coating. After entering the cell, NPs are transported to the cytoplasm and nucleus. The following are the mechanisms that cause toxicity, including cell cycle disorders, decreased mitochondrial function, reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) release, and triggering of apoptotic genes that lead to chromosomal aberrations, micronucleus formation, and DNA damage. BNPs can improve the efficiency of cancer treatment by reducing the adverse effects on normal cells by changing the nano surface. 21 BNPs showed a relative toxic potential to the KB cell line despite the use of low-dose concentrations. The hydrophilic PEG surface coating can help BNP to accumulate in cells, leading to cell pinocytosis; due to the electrostatic repulsion of negative charges, these negatively charged BNPs may penetrate through the cell membrane faster. 57 Once BNP enters the cell, it divides and releases silver, resulting in greater toxicity. Generally, solutions containing Ag-NPs and zero-valent silver (Ag°) sometimes appear in the form of ionic Ag. This is due to partial reduction of the precursor or oxidation of silver NPs to release Ag ions. When comparing Ag0 with cationic silver (Ag), the potential toxicity can be seen in the ionic form of silver. The toxicological efficacy varies with the oxidation state and the solubility characteristics of NPs. 58 Many types of monometallic gold-based radiosensitizers with shape and surface modification show a series of SER values. The SER value of gold nanorods is 1.21, which is the smallest TAT-Au NPs compared to 2.30. It is also worth noting that the SER of bismuth-based NP ranges from 1.06 to 1.53. Finally, tungsten and tantalum pass NP, showing values ​​of 1.22 and 1.33, respectively. 74,75 In silver-doped lanthanum manganese nanoparticles, with the increase of the silver component and the increase of silver ions, higher intracellular reactive oxygen species (ROS) and radiotoxicity were observed. The structure of NPs. 59 When KB cells were exposed to X-ray irradiation alone, a dose-dependent cell viability was observed relative to control cells; see Figure 10B. Dose-dependent results and significant differences between the different dose groups were observed (2 vs. 4Gy, p=0.2; 2 vs. 8Gy, p=0.04; 4 vs. 8Gy, p=0.46). It can be seen that the minimum dose required for cytotoxicity is 4Gy. In addition, previous studies have shown that molecular blockade can enhance radiosensitivity when irradiated with 4Gy. Therefore, the X-ray dose of 4Gy was chosen for subsequent experiments. 60 When cancer cells are exposed to ionizing radiation, the nucleus is damaged, the typical double-strand breaks, leading to the traditional "programmed cell death", that is, cell apoptosis. The main mechanism of cell loss of viability is radiation damage caused by internal mechanisms (such as DNA damage sensors, stress response, signaling pathways initiated by cytokines and ROS/RNS) or external mechanisms (such as upregulation of death receptors). Radiation-induced apoptosis can be mediated by the up-regulation and participation of death receptors such as TRAIL, Fas ligand (FasL), and programmed death ligand 1 (PDL1) or their corresponding receptors. The dose-dependent apoptosis induced by 2.5-10 Gy ionizing radiation increased the expression of p53, Fas, TRAIL, TRAIL-R2 and the down-regulation of Survivin anti-apoptotic protein in neuroblastoma stem cells. 61 Figure 10 (A) After treating KB cells with different doses of 50nm BNP for 24 hours, MTT was used for cell viability determination, and the KB cells were treated with different doses of 50nm BNP for 24 hours, and exposed to 4Gy X-ray doses (B) with different doses. Dose X-treated KB cell rays (0, 2, 4, 6 and 8 Gy). Cell viability is expressed as% cell viability ± SD (n = 3).

Figure 10 (A) KB cells were treated with different doses of 50nm BNP for 24 hours and MTT was used for cell viability determination. KB cells were treated with different doses of 50nm BNP for 24 hours and exposed to 4Gy X-ray dose (B) KB treated with KB X-rays of cells in different doses (0, 2, 4, 6 and 8 Gy). Cell viability is expressed as% cell viability ± SD (n = 3).

KB cells were treated with the same six low concentrations of 50nm BNP and incubated for 24 hours. After incubating for 24 hours, wash the 96-well plate with PBS, then add fresh medium, and then irradiate with 4Gy X-ray. Only internalized nanoparticles can cause radiosensitization. Compared with 4Gy radiation alone, the combination of 0.34 µg/mL BNP and 4Gy radiation significantly reduced the total cell survival (Figure 10A). In order for radiosensitization to be effective, BNP must undergo pinocytosis. 62 Compared with a lower dose, a higher concentration means more particle internalization. The results of this in vitro study show that even at smaller doses, BNP is an effective radiosensitizer. Compared with GNP alone, glucose-coated gold nanoparticles showed a 1.5-2.0-fold increase in growth inhibition; although GNP has been shown to significantly improve radiosensitivity in prostate cancer treatment. 63 BNP contains gold as one of the ingredients. The basic principle of its radiosensitivity is that compared with other size nanoparticles, the cell uptake rate of gold nanoparticles is high, so the viability of cells with internalized 50 nanometer gold nanoparticles will decrease under all irradiation. 11 In addition, it also shows that 50nm bimetallic Au-Ag NPs and Ag NPs are good radiosensitizers. 12 As mentioned above, Ag ion leakage is fatal. All these factors enhance the radiosensitivity of BNP. The synergy is due to the presence of high-Z element gold, metal ions release silver as Ag ions, the size of the nanoparticles is 50nm, and the final clinically significant dose is 4Gy. The sensitization enhancement rate (SER) for each BNP dose was calculated as the ratio between the cell viability of 4Gy exposed KB cells. Compared with 4Gy alone, BNP showed a 1.5-1.7-fold increase in growth inhibition. After demonstrating the radiosensitization potential of BNP under 4Gy X-ray radiation, we next examined the nuclear damage of KB cells under the same dose and X-ray radiation.

In order to evaluate the nuclear damage of KB cells treated with BNP 0.34 μg/mL, the synergistic effect of 4Gy X-ray radiation and (BNP 0.34 μg/mL 4Gy X-ray) radiosensitization was analyzed. Here, the positive control H2O2 used is 50μM/mL. The KB cells that received medium only were kept as a control for comparison. Apoptosis is determined by the presence of apoptotic bodies or chromatin condensation (ie, condensed and broken nuclei). The mitotic catastrophe is characterized by the presence of nuclei with two or more different lobes and micronuclei. Radiation-induced apoptosis is considered to be one of the main mechanisms of cell death after exposure to radiation. 64 Another study showed that mitotic disasters are considered to be the main mode of cell death induced by ionizing radiation, which is widely different from other clonal cell death mechanisms by well-known figures in the field of radiobiology. In addition, the catastrophe of apoptosis and mitosis may overlap to some extent. 36,65 As shown in Figure 11, the untreated control KB cells had normal nuclei, and no other obvious changes in cancer cell nuclei were observed. The cells treated with BNP showed mild apoptosis. KB and untreated Compared with the control cells, the radiation dose of 4Gy and 6 MV (without BNP) has a more destructive effect on the fragmented nuclei and chromatin condensed nuclei. Compared with the positive control without radiation dose, the highest nuclear damage was observed in the cells treated with 4Gy radiation of BNPs. Therefore, in this nuclear damage (apoptosis) study, it is concluded that BNPs can improve the curative effect of radiotherapy on KB cells, and can effectively prevent and inhibit the growth of KB cells. The percentages of all doses are shown in Figure 12. Figure 11 Confocal microscopy images of Hoechst stained nuclei of cells treated with BNP and X-rays. KB cells were irradiated with 4Gy X-rays, and the positive control H2O2 used was 50μM/mL. After 24 hours, the cells were fixed and stained with Hoechst. The imaging of the nucleus is done under the magnification of 20X and 60X lenses. Arrow: apoptosis; box: mitotic catastrophe. Scale bar = 60 μm. Figure 12 Quantitative data of nuclear apoptosis and mitotic disasters in KB cells treated with BNP and 4Gy X-rays. KB cells were irradiated with 4Gy X-rays, and the positive control H2O2 was 50μM/mL. After 24 hours, the cells were fixed and stained with Hoechst. The imaging of the nucleus is done under the magnification of 20X and 60X lenses. A total of 350 cell nuclei from random fields were evaluated for apoptosis and mitotic catastrophe (n=3).

Figure 11 Confocal microscopy images of Hoechst stained nuclei of cells treated with BNP and X-rays. KB cells were irradiated with 4Gy X-rays, and the positive control H2O2 used was 50μM/mL. After 24 hours, the cells were fixed and stained with Hoechst. The imaging of the nucleus is done under the magnification of 20X and 60X lenses. Arrow: apoptosis; box: mitotic catastrophe. Scale bar = 60 μm.

Figure 12 Quantitative data of nuclear apoptosis and mitotic disasters in KB cells treated with BNP and 4Gy X-rays. KB cells were irradiated with 4Gy X-rays, and the positive control H2O2 was 50μM/mL. After 24 hours, the cells were fixed and stained with Hoechst. The imaging of the nucleus is done under the magnification of 20X and 60X lenses. A total of 350 cell nuclei from random fields were evaluated for apoptosis and mitotic catastrophe (n=3).

In order to evaluate the ability of BNP as a CT nanocontrast agent, a clinical CT scanner was used to image the sample at a tube voltage of 120 kVp with a scan time of 3.0 seconds. X-ray absorption studies of BNP have been carried out with Omnipaque, which is often used to enhance CT contrast. Figure 13A depicts phantom model images at 5 mg/mL and 10 mg/mL obtained for Milli Q, 1x PBS, 0.6 mg/mL BNP, Omnipaque. The Hounsfield Unit (HU) value is calculated based on the selected region of interest (ROI) of a single sample. Figure 13B shows that Milli Q and 1X PBS have no contrast capacity; 0.6 mg/mL BNP measured 38 HU, while the X-ray absorption of 5 mg/mL and 10 mg/mL iodine in Omnipaque showed 120 and 220 HU, respectively. Although iodinated aromatic compounds are clinically injected into patients, they lack ideal characteristics due to the low X-ray attenuation when using high-kVp CT parameters. Give a relatively large dose of Omnipaque to obtain the best image quality. 66 The higher BNP seen by the concentration has a lower HU value, which is mainly due to the superposition and synergistic effect of the high X-ray attenuation coefficients of Au and Ag. The synthetic concentration is lower than that of iodine-based contrast agents. This corresponds to the X-ray attenuation ability of BNP that is nearly 4 times higher than that of iodine-based contrast agents, which may be attributed to the presence of electron-dense nano-gold and nano-silver particles in BNP. Compared with iodine (53 and 4.9 g/cm3, respectively), gold has a higher atomic number and electron density (79 and 19.32 g/cm3, respectively), suggesting this effect. It has been shown that at a concentration of 5 mg/mL, the CT signal intensity of naked nano Au is 354 HU, which is nearly 2 times higher than that of the mixed sample with the highest concentration of NPs. 67,68 Silver In dual-energy (DE) X-ray breast imaging, NP can be used as a potential substitute for iodine. Compared with iodine-based contrast agents, silver performs better; silver contrast agents are more beneficial than iodine, even if they are used clinically without changing the imaging system or protocol. 69 PEGylation can significantly improve the imaging quality of nanomaterials; it can also be seen that through dextran-coated SPIO and ultra-small SPIO (USPIO), extending t½ by 200 minutes can improve the image quality. 70 Figure 13 (A) CT image and (B) Milli Q, 1X PBS HU value, BNP 0.6mg/mL, Omnipaque 5mg/mL and Omnipaque 10mg/mL (n=3).

Figure 13 (A) CT image and (B) HU value of Milli Q, 1X PBS, BNP 0.6mg/mL, Omnipaque 5mg/mL and Omnipaque 10mg/mL (n=3).

The purpose of synthesizing BNP is to use them in a biological environment. In order to evaluate the stability of BNP, FBS was used as a biological culture medium, and turbidity determination was performed by measuring the turbidity change of the BNP suspension in FBS. Since the turbidity changes of the two suspensions were similar after 72 hours of incubation, no significant difference in turbidity between BNP and the control suspension was detected at different incubation times (Figure 14). No serum protein interaction or any aggregation was observed. Any unnecessary interactions between BNPs and serum proteins in the culture medium may lead to aggregation and loss of therapeutic efficacy and efficiency. The above data indicates that the synthesized BNPs will show excellent bioavailability in clinical scenarios. The negative control FBS combined with 5% glucose showed an increase in OD readings at 415nm, a possible explanation for this is exponential bacterial growth. Figure 14 The stability of BNPs in fetal bovine serum. Negative control: use 5% glucose.

Figure 14 The stability of BNPs in fetal bovine serum. Negative control: use 5% glucose.

This study reported for the first time the one-pot synthesis of PEGylated bimetallic Au-Ag NPs using PEG 600. The orange-brown well-dispersed colloidal BNPs PEG-terminated nanostructures are stable and more active, have a uniform size distribution, have an average hydrodynamic size of ~50±5nm, are negatively charged, and have a zeta potential of about -5mV. The electron microscope showed its nearly spherical morphology, monodisperse uniform diameter and 35nm size. HAADF-STEM and elemental mapping studies have shown that Au and Ag are the constituent metals of the nano-alloy mixture. 1H NMR spectroscopy shows the extensive participation of -OH in the synthesis. When BNP is exposed to oral cancer cells, they effectively enter the cell into the cytoplasm and the nuclear periphery, resulting in enhanced radiation effects, as shown by in vitro radiosensitization, the enhancement rate is about 1.5-1.7. In summary, the current study confirmed the radiosensitization effect of BNPs on KB cells (oral cancer) under 4Gy X-ray irradiation. Nuclear imaging with Hoechst staining showed that the strong apoptotic effect of internalized BNP resulted in irreversible DNA damage. Compared with the clinically used Omnipaque agent, BNP shows relatively higher CT contrast ability. In addition, BNP is stable in a biological environment with the best bioavailability and will not reunite. The synergy of the constituent materials greatly promotes the potential applications of nano-alloy materials. 71 Further research is needed to verify the radiosensitization effect on large tumors in in vivo studies, and to evaluate the molecular mechanism through mRNA expression. Finally, nanotoxicology research and biodistribution will help to understand more about the potential of BNP in nanotherapy. The results obtained in this study are promising. We are currently investigating BNP loaded with doxorubicin, which is an enhanced nano-drug for combined treatment of oral cancer with radiotherapy and chemotherapy strategies.

This work will be included in the doctoral dissertation: "Radiosensitizer and drug delivery system based on gold nanoparticles." In addition, one of the authors, Shameer Ahmed B, would like to thank BITS Pilani KK Birla Goa Campus, Goa for providing institutional scholarships for his PhD term. All authors would like to thank the DLS and Raman spectroscopy of the University of Mysore, the ICPMS of IIT (Indian Institute of Technology) Delhi, the Center for Nanoscience and Engineering (CeNSE)-IISc (Indian Institute of Science), HRTEM in Bangalore, HAADF-STEM, EDX. Manipal Hospital, Goa provides LINAC irradiation and CT scanning equipment, as well as Professor Vinod K. Aswal, Department of Solid State Physics, Bhabha Atomic Research Center, Trombay, Mumbai, responsible for the design, measurement and analysis of SAXS and SANS. Finally, we thank Dr. Varsha. K. Pavithran, Rajah Muthiah School of Dentistry and Hospital, Anna Marai University, assisted in the statistical analysis of this research article.

All authors have made significant contributions to the work of the report, whether in terms of concept, research design, execution, data acquisition, analysis and interpretation, or in all these areas; participating in drafting, revising or critically reviewing articles; final approval requirements Published version; agreed on the journal to which the article was submitted; and agreed to be responsible for all aspects of the work.

This research will be a chapter of Mr. Shameer Ahmed's paper and will be uploaded to the Birla Institute of Technology and Science repository in 2022. The authors declare that they have no competing interests.

1. Tshering Vogel DW, Zbaeren P, Thoeny HC. Oral cancer and oropharyngeal cancer. Cancer imaging. 2010;10(1):62–72. doi:10.1102/1470-7330.2010.0008

2. Ketabat F, Pundir M, Mohabatpour F, etc. Controlled drug delivery system for oral cancer treatment-current status and future prospects. pharmaceutics. 2019;11(7):302. doi:10.3390/pharmaceutics11070302

3. Vanetti E, Clivio A, Nicolini G, etc. Volume modulated arc radiation therapy for oropharyngeal cancer, hypopharyngeal cancer and laryngeal cancer: comparison with the treatment plan of fixed-field IMRT. Radiother Oncol. 2009;92(1):111–117. doi:10.1016/j.radonc.2008.12.008

4. Brun E, Sicard-Roselli C. Practical problems caused by nanoparticle radiosensitization. Radiat Phys Chem Oxf Engl. 2016; 128: 134-142. doi:10.1016/j.radphyschem.2016.05.024

5. Wang Hua, Mu X, He Hua, Zhang Xiaodong. Cancer radiosensitizer. Trends in pharmacology. 2018;39(1):24–48. doi:10.1016/

6. Zhang Xiaodong, Luo Zhi, Chen Jie, etc. Ultra-small Au(10-12)(SG)(10-12) nanomolecules for high tumor specificity and cancer radiotherapy. Adv Mater. 2014;26(26):4565-4568. doi:10.1002/adma.201400866

7. Liu Ping, Jin Hua, Guo Zhi, etc. In vitro and intracranial glioma mouse models, silver nanoparticles performed better than gold nanoparticles in radiosensitized U251 cells. International J Nanomedicine. 2016; 11:5003-5014. doi:10.2147/IJN.S115473

8. Zhao Jian, Liu Ping, Ma Jian, etc. Silver nanoparticles functionalized with polyethylene glycol and aptamer As1411 enhance radiosensitization for radiotherapy of glioma. International J Nanomedicine. 2019; 14: 9483–9496. doi:10.2147/IJN.S224160

9. Maggiorella L, Barouch G, Devaux C, etc. Use hafnium oxide nanoparticles for nano-level radiation therapy. Future tumors. 2012; 8(9): 1167–1181. doi:10.2217/fon.12.96

10. Hoth, MCA, Huang Kun, etc. Excellent penetration and retention behavior of 50 nm gold nanoparticles in tumors. Cancer research. 2013;73(1):319-330. doi:10.1158/0008-5472.CAN-12-2071

11. Chithrani DB, Jelveh S, Jalali F, etc. Gold nanoparticles are used as radiosensitizers in cancer treatment. Radiation Reservoir 2010;173(6):719-728. doi:10.1667/RR1984.1

12. Fat MM. Thymoquinone was used to biosynthesize silver nanoparticles and evaluate its radiosensitizing activity. Bio-Nanoscience. 2020;10(1):260–266. doi:10.1007/s12668-019-00702-3

13. Le Tourneau C, Calugaru V, Thariat JO, etc. Hafnium oxide nanoparticles are a promising new treatment method for head and neck cancer. Int J Radiat Oncol Biol Phys. 2018;100(5):1377. doi:10.1016/j.ijrobp.2017.12.180

14. Sreekumaran Nair A, Suryanarayanan V, Pradeep T, Thomas J, Anija M, Philip R. [Email protection] Core-shell nanoparticles: synthesis, characterization, reactivity, and optical limitations. Mater Sci Eng B Solid State Mater Adv Technol. 2005;117(2):173-182. doi:10.1016/j.mseb.2004.11.010

15. Shim K, Lee WC, Heo YU, etc. Reasonably designed bimetal [email protection] nanoparticles are used for glucose oxidation. Sci Rep. 2019;9(1):894. doi:10.1038/s41598-018-36759-5

16. Cui L, Her S, Borst GR, Bristow RG, Jaffray DA, Allen C. Radiosensitization of gold nanoparticles: Will they enter the clinic? Radiother Oncol. 2017;124(3):344–356. doi:10.1016/j.radonc.2017.07.007

17. Generalov R, Kuan WB, Chen W, Kristensen S, Juzenas P. The radiosensitization effect of zinc oxide and silicon dioxide nanocomposites on cancer cells. Colloidal surfing B Biological interface. 2015; 129: 79-86. doi:10.1016/j.colsurfb.2015.03.026

18. Molina Higgins MC, Clifford DM, Rojas JV. [Email protection] Nanocomposites synthesized by X-ray radiation decomposition are used as potential radiosensitizers. Applied surfing science. 2018; 427: 702-710. doi:10.1016/j.apsusc.2017.08.094

19. Her S, Jaffray DA, Allen C. Application of gold nanoparticles in cancer radiotherapy: mechanism and latest developments. Adv Drug Deliv Rev. 2017; 109:84-101. doi:10.1016/j.addr.2015.12.012

20. Zhang Xindong, Wu De, Shen X, Liu PX, Fan FY, Fan SJ. Kidney clearance, biodistribution, and toxicity of gold nanoclusters in vivo. biomaterials. 2012;33(18):4628-4638. doi:10.1016/j.biomaterials.2012.03.020

21. Botha TL, Elemike EE, Horn S, Onwudiwe DC, Giesy JP, Wepener V. Cytotoxicity of Ag, Au and Ag-Au bimetallic nanoparticles prepared using gold rod (Solidago canadensis) plant extract. Sci Rep. 2019;9(1):4169. doi:10.1038/s41598-019-40816-y

22. Lomelí-Marroquín D, Medina Cruz D, Nieto-Argüello A, etc. Starch-mediated synthesis of monometallic and bimetallic silver/gold nanoparticles are used as antibacterial and anticancer agents. International J Nanomedicine. 2019;14:2171-2190. doi:10.2147/IJN.S192757

23. Mukha I, Vityuk N, Grodzyuk G, etc. Anticancer effect of Ag, Au and Ag/Au bimetallic nanoparticles prepared in the presence of tryptophan. J Nanosci Nanotechnology. 2017;17(12):8987–8994. doi:10.1166/jnn.2017.14106

24. Katifelis H, Lyberopoulou A, Mukha I, etc. Ag/Au bimetallic nanoparticles induce apoptosis in human cancer cell lines through the P53, CASPASE-3 and BAX/BCL-2 pathways. Artif Cell Nanomedicine Biotechnology. 2018;46(sup3):S389–S398. doi:10.1080/21691401.2018.1495645

25. Ritschel T, Lehmann K, Brunzel M, etc. The well-defined poly(ethylene glycol) polymer is used as an unconventional reaction tracer for colloidal transport in porous media. J Colloid Interface Science. 2021; 584: 592-601. doi:10.1016/j.jcis.2020.09.056

26. Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R. Biodegradable long-circulation polymer nanospheres. science. 1994;263(5153):1600-1603. doi:10.1126/science.8128245

27. Shkilnyy A, Soucé M, Dubois P, Warmont F, Sabongi ML, Chourpa I. Poly(ethylene glycol) stabilized silver nanoparticles are used in biological analysis applications for SERS spectroscopy. Analyst. 2009;134(9):1868-1872. doi:10.1039/b905694g

28. Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. Pegylation is used as a strategy to improve nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2016;99(Pt A):28-51. doi:10.1016/j.addr.2015.09.012

29. Kwatra D, Venugopal A, Anant S. Nanoparticles in radiotherapy: a summary of various methods for enhancing cancer radiosensitization. Translate cancer research. 2013; 2(4). doi:10.3978/j.issn.2218-676X.2013.08.06

30. Wang M, Thanou M. Targeting nanoparticles to cancer. Pharmaceutical research. 2010;62(2):90–99. doi:10.1016/j.phrs.2010.03.005

31. Zopes D, Hegemann C, Schläfer J, Tyrra W, Mathur S. Single-source precursor of alloy gold and silver nanocrystals-a molecular metallurgical method. Inorganic Chemistry 2015; 54(8): 3781-3787. doi:10.1021/ic502924s

32. Ristig S, Kozlova D, Meyer-Zaika W, Epple M. Simple synthesis of autofluorescent alloy silver-gold nanoparticles. J Mater Chem B Mater Biol Med. 2014; 2(45): 7887-7895. doi:10.1039/c4tb01010h

33. Soulé S, Bulteau AL, Faucher S, etc. Biomimetic Au-Ag [email protection] The design and cell fate of silica nanoparticles. Langmuir. 2016;32(39):10073–10082. doi:10.1021/acs.langmuir.6b02810

34. Naha PC, Lau KC, Hsu JC, etc. Gold-silver alloy nanoparticles (GSAN): An imaging probe for breast cancer screening, used in dual-energy mammography or computed tomography. nanoscale. 2016;8(28):13740–13754. doi:10.1039/c6nr02618d

35. Ahmed BS, Rao AG, Sankarshan BM, etc. Evaluation of gold, silver and silver-gold (bimetallic) nanoparticles as radiosensitizers in radiotherapy in cancer treatment. Cancer Tumor Research Center. 2016;4(3):42–51. doi:10.13189/cor.2016.040302

36. Kobayashi D, Shibata A, Oike T, Nakano T. A one-step protocol for evaluating radiation-induced clonal cell death patterns by fluorescence microscopy. J Vis Exp. 2017; 128. doi: 10.3791/56338.

37. Li Tao, Shen X, Chen Y, et al. Polyetherimide grafted Fe3O4@SiO22 nanoparticles can be used as a therapeutic and diagnostic agent for simultaneous VEGF siRNA delivery and magnetic resonance cell imaging. International J Nanomedicine. 2015; 10: 4279-4291. doi:10.2147/IJN.S85095

38. Sharma C, Ansari S, Ansari MS, Satsangee SP, Srivastava MM. Use lilac flower bud extract to synthesize Au/Ag bimetallic nanoparticles in one step: enhance anti-oxidant biological efficacy and catalytic activity. Mater Sci Eng C Mater Biol Appl. 2020;116(111153):111153. doi:10.1016/j.msec.2020.111153

39. Stiufiuc R, Iacovita C, Nicoara R, etc. One-step synthesis of PEGylated gold nanoparticles with adjustable surface charge. J nano materials. 2013; 2013: 1-7. doi:10.1155/2013/146031

40. Alam MJ, Tsuji M, Matsunaga M, Yamaguchi D. Under oil bath heating, the shape of the Au-Ag bimetallic system changes, from polygonal Au nanocrystals to spherical Au/Ag alloys and eccentric Au core Ag/Au alloy shell particles. CrystEngComm. 2011;13(8):2984–2993. doi:10.1039/c0ce00899k

41. Huang Jian, Li Qiang, Sun De, etc. A new type of camphor stem leaf biosynthesis of silver and gold nanoparticles. nanotechnology. 2007;18(10):105104. doi:10.1088/0957-4484/18/10/105104

42. Laaksonen T, Ahonen P, Johans C, Kontturi K. Stability and electrostatics of mercaptoundecanoic acid terminated gold nanoparticles with different counterion sizes. Chemical physics. 2006;7(10):2143–2149. doi:10.1002/cphc.200600307

43. Patsula V, Horák D, Kučka J, etc. The synthesis and modification of homogeneous PEG-neridronate modified magnetic nanoparticles determines the prolonged blood circulation and biodistribution in the mouse preclinical model. Sci Rep. 2019;9(1):10765. doi:10.1038/s41598-019-47262-w

44. Zhang H, Okuni J, Toshima N. One-pot synthesis of Ag-Au bimetallic nanoparticles with Au shells and their high catalytic activity for aerobic glucose oxidation. J Colloid Interface Science. 2011;354(1):131–138. doi:10.1016/j.jcis.2010.10.036

45. Murugavelu M, Karthikeyan B. Synthesis and characterization of Ag-Au core-shell bimetallic nanoparticles and their application in electrocatalytic oxidation/sensing of l-methionine. Mater Sci Eng C Mater Biol Appl. 2017; 70 (Part 1): 656–664. doi:10.1016/j.msec.2016.09.046

46. ​​Banerjee M, Sharma S, Chattopadhyay A, Ghosh SS. The antibacterial activity of bimetallic gold-silver core-shell nanoparticles is enhanced at low silver concentrations. nanoscale. 2011;3(12):5120-5125. doi:10.1039/c1nr10703h

47. Vinod M, Gopchandran KG. Au, Ag and Au: Ag colloidal nanoparticles synthesized by pulsed laser ablation are used as SERS substrates. Prog Nat Sci. 2014; 24(6): 569–578. doi:10.1016/j.pnsc.2014.10.003

48. Berahim N, Basirun W, Leo B, Johan M. Synthesis of bimetallic gold-silver (Au-Ag) nanoparticles for catalytic reduction of 4-nitrophenol to 4-aminophenol. catalyst. 2018;8(10):412. doi:10.3390/catal8100412

49. Guadagnini A, Agnoli S, Badocco D, etc. Easily synthesize non-equilibrium cobalt-silver nanoparticles with magnetic and plasma properties in liquid by laser ablation. J Colloid Interface Science. 2021; 585: 267-275. doi:10.1016/j.jcis.2020.11.089

50. Pinzaru I, Coricovac D, Dehelean C, etc. Stable PEG-coated silver nanoparticles-comprehensive toxicological characteristics. Food Chemical Toxicology. 2018; 111: 546-556. doi:10.1016/j.fct.2017.11.051

51. Vodnik VV, Mojić M, Stamenović U, etc. The development of genistein-loaded gold nanoparticles and their anti-tumor potential for prostate cancer cell lines. Mater Sci Eng C Mater Biol Appl. 2021;124(112078):112078. doi:10.1016/j.msec.2021.112078

52. Tian F, Bonnier F, Casey A, Shanahan AE, Byrne HJ. Surface enhanced Raman scattering of gold nanoparticles: the effect of particle shape. Anal method. 2014; 6(22): 9116-9123. doi:10.1039/c4ay02112f

53. Dai Li, Song Li, Huang Y, etc. A bimetallic Au/Ag core-shell superstructure with tunable surface plasmon resonance and high-performance surface-enhanced Raman scattering in the near-infrared region. Langmuir. 2017;33(22):5378–5384. doi:10.1021/acs.langmuir.7b00097

54. Dust JM, Fang ZH, Harris JM. Proton NMR characterization of poly(ethylene glycol) and derivatives. Macromolecule. 1990;23(16):3742–3746. doi:10.1021/ma00218a005

55. Xu X, He Z, Lu S, Guo D, Yu J. Use reactive compatibilizers containing flexible segments to improve the thermal and mechanical properties of lignin/polypropylene wood-plastic composites. Macromolecule Reservoir 2014;22(10):1084-1089. doi:10.1007/s13233-014-2161-3

56. Meabe L, Sardon H, Mecerreyes D. Hydrolytically degradable poly(ethylene glycol)-based polycarbonate through organic catalytic condensation. Eur Polym J. 2017; 95: 737-745. doi:10.1016/j.eurpolymj.2017.06.046

57. Sharifi F, Jahangiri M, Nazir I, etc. Zeta potential change nanoemulsion based on simple zwitterion. J Colloid Interface Science. 2021;585:126-137. doi:10.1016/j.jcis.2020.11.054

58. Repair ZM, Zhang QB, Puppala HL, Colvin VL, Alvarez PJJ. The particle-specific antibacterial activity of silver nanoparticles is negligible. Nanolet. 2012; 12(8): 4271-4275. doi:10.1021/nl301934w

59. Khochaiche A, Westlake M, O'Keefe A, etc. It is the first extensive study of silver-doped lanthanum manganate nanoparticles to induce selective chemotherapy and enhance radiotoxicity. Mater Sci Eng C Mater Biol Appl. 2021;123(111970):111970. doi:10.1016/j.msec.2021.111970

60. Teraoka S, Kakei Y, Akashi M, etc. Gold nanoparticles enhance X-ray radiation-induced apoptosis of head and neck squamous cell carcinoma cells in vitro. Biomedical Representative 2018; 9(5): 415–420. doi:10.3892/br.2018.1142

61. McKelvey KJ, Hudson AL, Back M, Eade T, Diakos CI. Radiation, inflammation and immune response in cancer. Mother genome. 2018;29(11–12):843–865. doi:10.1007/s00335-018-9777-0

62. Bhattarai SR, Derry PJ, Aziz K, etc. Gold nano triangles: magnification and X-ray radiation sensitization effects in mice. nanoscale. 2017; 9(16): 5085–5093. doi:10.1039/c6nr08172j

63. Zhang X, Xing Jianzhong, Chen Jie, etc. Enhance the radiation sensitivity of prostate cancer through gold nanoparticles. Clinical investment medicine. 2008; 31(3): E160-7. doi:10.25011/cim.v31i3.3473

64. Eriksson D, Stigbrand T. The mechanism of cell death induced by radiation. Tumor biology. 2010;31(4):363–372. doi:10.1007/s13277-010-0042-8

65. Waters BG. Cell death after radiation: how, when and why cells die. In: Basic Clinical Radiobiology. CRC Press; 2018: 21-31. doi:10.1201/9780429490606-3

66. Meenambal R, Kannan S. Design and structure study of Yb3 substituted β-Ca3(PO4)2 contrast agent for bimodal NIR luminescence and X-ray CT imaging. Mater Sci Eng C Mater Biol Appl. 2018; 91: 817-823. doi:10.1016/j.msec.2018.06.032

67. Mishra SK, Kannan S. Adriamycin-coupled bimetallic silver-gadolinium nano-alloy for multi-modal MRI-CT optical imaging and pH-responsive drug release. ACS Biomaterials Science Engineering. 2017; 3(12): 3607-3619. doi:10.1021/acsbiomaterials.7b00498

68. Narayanan S, Sathy BN, Mony U, Koyakutty M, Nair SV, Menon D. A biocompatible magnetite/gold nano hybrid contrast agent for MRI and CT bioimaging through green chemistry. ACS application program interface. 2012; 4(1): 251–260. doi:10.1021/am201311c

69. Karunamuni R, Tsourkas A, Maidment AD. Explore silver as a contrast agent to enhance dual-energy X-ray breast imaging. Br J Radiol. 2014;87(1041):20140081. doi:10.1259/bjr.20140081

70. Jokerst JV, Lobovkina T, Zare RN, Gambhir SS. Pegylation of nanoparticles for imaging and therapy. Nanomedicine (London). 2011; 6(4): 715–728. doi:10.2217/nnm.11.19

71. Rachna RM, Shanker U. The synergistic effect of zinc oxide coupled copper hexacyanoferrate nanocomposites: powerful visible light drives dye degradation. J Colloid Interface Science. 2021; 584: 67-79. doi:10.1016/j.jcis.2020.09.079

72. Bindhu J, Anupama G. Radiosensitization: a new dogma in cancer treatment. Australia-Asia J cancer. 2005;4(4):241-50. ISSN-0972-2556.

73. Xiong Z, Qin F, Huang PS, Nettleship I, Lee JK. The influence of synthesis technology on the crystallization and optical properties of Ag-Cu bimetallic nanoparticles. Chaom. 2016;68(4):1163–1168. doi:10.1007/s11837-015-1757-1

74. Ma Nan, Fugen W, Zhang X, et al. Shape-dependent radiosensitization effects of gold nanostructures in cancer radiotherapy: comparison of gold nanoparticles, nanopins and nanorods. ACS application program interface. 2017; 9(15): 13037–13048. doi:10.1021/acsami.7b01112

75. Zhang X, Chen X, Jiang Yongwei, etc. Glutathione depletion gold nanoclusters enhance cancer radiotherapy through coordinated external and internal regulation. ACS application program interface. 2018;10(13):10601–10606. doi:10.1021/acsami.8b00207

76. Ma N, Liu P, Nongyue H, Ning G, Fu-Gen W, Chen Z. The role of nano-radiosensitizers based on gold nano-nails: cell internalization, radiotherapy and autophagy. ACS application program interface. 2017;9(37):31526–31542. doi:10.1021/acsami.7b09599

77. Rawat L, Hegde H, Hoti SL, Nayak V. Piperlongumine induces ROS-mediated cell death in human intestinal cancer cells and cooperates with paclitaxel. Biomedical pharmaceutical company. 2020; 128: 110243–110249. doi:10.1016/j.biopha.2020.110243

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and include the Creative Commons Attribution-Non-commercial (unported, v3.0) license. By accessing the work, you hereby accept the terms. The use of the work for non-commercial purposes is permitted without any further permission from Dove Medical Press Limited, provided that the work has an appropriate attribution. For permission to use this work for commercial purposes, please refer to paragraphs 4.2 and 5 of our terms.

Contact Us• Privacy Policy• Associations and Partners• Testimonials• Terms and Conditions• Recommend this site• Top

Contact Us• Privacy Policy

© Copyright 2021 • Dove Press Ltd • Software development of • Web design of Adhesion

The views expressed in all articles published here are those of specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.

Dove Medical Press is part of Taylor & Francis Group, the academic publishing department of Informa PLC. Copyright 2017 Informa PLC. all rights reserved. This website is owned and operated by Informa PLC ("Informa"), and its registered office address is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 3099067. UK VAT group: GB 365 4626 36

In order to provide our website visitors and registered users with services that suit their personal preferences, we use cookies to analyze visitor traffic and personalize content. You can understand our use of cookies by reading our privacy policy. We also retain data about visitors and registered users for internal purposes and to share information with our business partners. By reading our privacy policy, you can understand what data we retain, how we process it, who we share it with, and your right to delete data.

If you agree to our use of cookies and the content of our privacy policy, please click "Accept".