Development of Gold-Bismuth Sulfide Nanorods (Au@Bi2S3 NRs) for X-ray- based imaging

Development of Gold-Bismuth Sulfide Nanorods (Au@Bi2S3 NRs) for X-ray- based imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Gold-Bismuth Sulfide Nanorods (Au@Bi 2 S 3 NRs) for X-ray- based imaging Bhavesh D Kevadiya, Rosita Primavera, Ganesh Swaminathan, Jim Zhong, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8854864/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The development of hybrid metal nanoparticles (NPs) for use as computed tomography (CT) contrast agents is a promising area of research. Achieving optimal in vivo performance of imaging for NPs is challenging and depends on their geometry, materials properties, bioreactivity, and biocompatibility. In this study, we designed and developed a novel CT contrast agent composed of gold nanoparticles (AuNPs) coordinated on the surface of bismuth sulfide-core nanorods (Au@Bi 2 S 3 NRs) utilizing a solvothermal synthesis approach. We conducted solid-state characterization of Au@Bi 2 S 3 NRs, demonstrating their structural configuration, excellent stability, uniformity, and high crystallinity. We also tested their biocompatibility with mesenchymal stem cells and found they exhibited no toxic effects. To evaluate the imaging potential of Au@Bi 2 S 3 NRs, we tested them in small animals using CT imaging. Our results showed contrast enhancement in soft tissues, indicating the retention of the particles at these locations with no local inflammatory responses. Taken together, our study provides a proof-of-concept for the robust synthesis and use of Au@Bi 2 S 3 NRs as effective CT imaging contrast agents. Future work will explore the potential to functionalize Au@Bi 2 S 3 NRs with therapeutic molecules for theranostic applications. CT imaging Gold nanoparticles Bismuth sulfide nanorods Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Metal nanoparticles (NPs) have unique properties that make them ideal for bioimaging applications, but their potential toxicity must be carefully considered [ 1 , 2 ]. The high-energy blocking capabilities of metal NPs make them useful for computed tomography (CT) imaging [ 3 , 4 ]. CT is a widely employed imaging modality that utilizes X-rays to generate detailed three-dimensional images of the body, relying on the varying densities of tissues to block X-rays to different extents [ 5 – 7 ]. Despite its effectiveness, distinguishing between tissues with similar densities can be challenging due to subtle differences in X-ray absorption, which may complicate image interpretation. To address this issue, contrast agents are employed. These agents can either accumulate in specific tissues or fill luminal spaces, such as blood vessels or the gastrointestinal tract, thereby enhancing X-ray absorption and improving image contrast [ 8 ]. Commonly used agents, like iodine and barium, can increase the radiodensity of tissues within which they reside, and are generally considered safe [ 9 , 10 ], though they can be associated with adverse reactions, thyroid gland dysfunction, and nephropathy [ 11 – 13 ]. Additionally, the transit of these conventional contrast agents through tissues is relatively rapid, especially when administered via a vascular route; accordingly, precise timing is required for imaging or repeated CT dosing. However, these issues can potentially be improved by using electron-dense nanomaterials that can be retained longer in tissues [ 14 – 16 ]. In recent years, metal NPs have become increasingly utilized as medical imaging agents due to their improved biodistribution, biostability, predictable cellular interactions, and reproducible synthesis, especially compared to traditional contrast agents [ 1 , 2 , 4 , 17 , 18 ]. Indeed, gold (Au) and bismuth (Bi) based NPs have been well studied as potential contrast agents for CT imaging [ 10 , 19 – 21 , 35 – 39 ]. Spherical AuNPs have several advantages, including their high atomic number, high X-ray attenuation, non-toxicity, facile synthesis, and surface functionalization for colloidal stability [ 15 , 19 – 23 ]. Rod-shaped bismuth sulfide nanorods (Bi 2 S 3 NRs) also have strong X-ray absorption, excellent biocompatibility, high stability, and relatively long circulation times [ 4 , 10 , 24 – 26 ]. By combining the properties of both these NPs, hybrid bimetal NPs can be created, which may offer improved imaging performance compared to either type of metal NPs alone. The synthesis of both AuNPs and Bi 2 S 3 NRs is a challenging task, given their rapid oxidation, which decreases their stability and shelf life. Accordingly, these NPs require the use of hazardous chemicals and manufacturing processes with high production costs, which can limit their clinical translation. In the present study, we developed an approach to reproducibly make a hybrid metal gold-bismuth-sulfide particle (i.e. Au@Bi 2 S 3 NRs) of high quality, uniformity, and stability using a solvothermal synthesis approach. Here, spherical AuNPs were decorated onto the surface of Bi 2 S 3 NRs in situ, and then Au@Bi 2 S 3 NRs were bioanalytically characterized using state-of-the-art tools, followed by in vitro and in vivo biocompatibility testing and in vivo imaging using CT. 2. Experimental Section 2.1. Chemicals and Reagents Gold (III) chloride trihydrate (HAuCl 4 .3H 2 O), thioacetamide (CH 3 CSNH 2 ), Bismuth neodecanoate (Bi(OCOC(CH 3 ) 2 (CH 2 ) 5 CH 3 ) 3 ), sodium borohydride (NaBH 4 ), oleic acid (OA), oleylamine (OAm, 70%), and L-α-phosphatidylcholine (PC) (from egg yolk), were obtained from MilliporeSigma, St. Louis, MO, USA. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-fluorescein (sodium salt) (FITC-DSPE-PEG 2,000) was obtained from Xi'an ruixi Biological Technology Co, China. DSPE-PEG 2,000 (N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) was obtained from NOF Corporation, Tokyo, Japan. Human Wharton’s Jelly-derived mesenchymal stem cells (UC-MSCs) were obtained from StemBioSys, San Antonio, TX, USA. DAPI (4',6-diamidino-2-phenylindole) was obtained from Thermo Fisher Scientific, USA. Cell culture media MesenCult™ and human platelet lysate were obtained from Stemcell Technologies, Vancouver, BC, CA. CellTiter-Glo™ was purchased from Promega, Madison, WI, USA. Male rats were purchased from Charles River Laboratories, USA. 2.2. Synthesis of Au@Bi 2 S 3 NRs Core bismuth sulfide (Bi 2 S 3 ) nanorods (NRs) were synthesized using the solvothermal method [ 25 ] followed by decoration with AuNPs. Briefly, 1.45 g (~ 1.87 mmol) of bismuth neodecanoate and 20 mL of OA were placed into a 500 mL capacity Teflon-lined autoclave reactor and the mixture was stirred for 10 minutes. Subsequently, 10 mL of ethanol was added dropwise and mixed for 10 minutes at 300 rpm. In a separate glass vial, 150 mg of thioacetamide (~ 1.99 mmol) was mixed with 4 mL of OAm and sonicated until a uniform, light yellow-colored solution was obtained. The thioacetamide-OAm mixture was then transferred to a Teflon-lined stainless-steel autoclave, and the solution turned black-colored. This mixture was stirred for 1 h, placed in a sealed autoclave that was maintained at 150°C for 8 h, and then allowed to cool to room temperature. The precipitate was collected by centrifugation, washed several times with 200-proof ethanol, and dried overnight in a desiccator. The Bi 2 S 3 NRs were finally obtained as a black powder. The Bi 2 S 3 NRs were decorated with AuNPs using a seed-mediated growth method, which involved the following steps: Step one - Bi 2 S 3 NRs (25 mg) powder was dispersed in anhydrous chloroform (1 mL). DSPE-PEG 2000 (25 mg) was mixed in chloroform (1 mL) in a separate glass vial. Both components were bath sonicated to ensure they were completely dispersed and dissolved. Step two - the solutions were mixed with 2 mL of DI water, and the solvent was then evaporated slowly by heating at 80°C in a glass vial. Step three - the Bi 2 S 3 NRs obtained as a black homogenous mixture was dispersed in 50 mL of DI water and heated it at 80°C for 30 min while stirring at 500 rpm. Step four − 1 mL of a HAuCl 4 .3H 2 O (10 mmol) aqueous solution was added to Bi 2 S 3 -DSPE-PEG 2000 solution. After 20 min, a reducing agent, 600 µL of ice-cold NaBH 4 solution in DI water (1 mg/mL) was introduced to initiate the surface-confined decorating of uniform AuNPs while stirring for an additional 30 min at 95°C at 500 rpm. During this time, the colloidal solution changed in color from black to brownish-black, indicating the formation of AuNPs decorating Bi 2 S 3 NRs (Au@Bi 2 S 3 NRs). Step five - Au@Bi 2 S 3 NRs were further purified by centrifugation at 7,000 rpm for 30 min, washed with DI water and dispersed in DI water for further characterization. The resultant AuNPs were typically ~ 2–3 nm in diameter, and these can be easily tuned by modulating the amount of gold ions used. For cell and animal testing, lipid-coated Au@Bi 2 S 3 NRs were prepared using a thin-film dispersion method. Briefly, DSPE-PEG 2000 and PC (50:50% w/w) were dissolved in 5 mL of chloroform in a round-bottom flask to afford a thin film upon solvent evaporation, which was subsequently vacuum dried. Au@Bi 2 S 3 NRs (~ 20 mg), dispersed in cyclohexane and mixed with 1% (v/v) Tween-80, were sonicated. Cyclohexane was evaporated from the Au@Bi 2 S 3 NRs/cyclohexane/Tween-80 emulsion. A Tween-80-coated Au@Bi 2 S 3 NRs solution was next added to the lipid film flask and dispersed in a lipid film at 45°C with bath sonication. The product was collected after centrifugation at 500 rpm for 10 minutes and then dispersed in MilliQ water for further characterization and CT imaging testing. FITC-DSPE-PEG 2000 sodium salt was used as a fluorescence conjugate. The FITC-DSPE-PEG 2000 mass ratios were selected as 0.25% w/w with PC and DSPE-PEG2000 to form a lipid film for FITC-Au@Bi 2 S 3 NRs and to use for cell study. The remaining purification steps of the procedure were the same as above. 2.3. Characterization of Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs The morphology, lattice crystal structure, elemental composition, and chemical color mapping of the synthesized Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were determined by brightfield high-resolution transmission electron microscopy (HR-TEM), selected area electron diffraction (SAED), energy-dispersive X-ray (EDX) spectroscopy, and scanning transmission electron microscopy (STEM) with high-angle annular dark-field (HAADF) (FEI Tecnai Osiris S/TEM). The TEM samples were prepared in cyclohexane and dried on a copper grid (Electron Microscopy Sciences-EMS, Hatfield, PA, USA) at room temperature. The additional gain in speed can also be used to collect EDX elemental mappings from a larger field-of-view. The lattice fringes of the obtained samples and the corresponding SAED patterns were examined using HR-TEM at 200 kV. The experimental SAED patterns were analyzed using PCED2.0 software. The particles were also characterized by performing wavelength dispersive X-ray fluorescence (WDXRF) analysis using a Rigaku WDXRF (Supermini200) spectrometer with high resolution and lower detection limits for elemental analysis. A 200 W, air-cooled, Pd X-ray source was operated at 50 kV and 4 mA to produce excitation spectra with faster elemental detection capability. A three-crystal analyzing unit was equipped in the system to support the standard LiF (200). Nanoparticle surface chemistry analysis was performed with X-ray photoelectron spectroscopy (XPS) and measurements were carried out using monochromatic Al K-alpha (α) X-ray with an energy of 1486.6 eV by Thermo Scientific K-alpha + XPS (Thermo Fisher Scientific, Waltham, MA, USA). Powder X-ray diffraction (XRD) analyses of Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were performed in the 2θ range of 2–60° using a PANalytical Empyrean diffractometer (PANalytical Inc.; Westborough, MA, USA) with Cu-Kα radiation (1.5418 Å) at 40 kV, 45 mA settings. A mask of 20 mm and a divergence slit of 1/32° were used for the incident beam path. A thin layer of the nanoparticle powder sample was placed on a zero-background silicon plate and the sample holder then continuously spun at the rate of 22.5 deg/s during all measurements. The PIXcel3D detector, equipped with a beam monochromator (PANalytical Inc.; Westborough, MA, USA) was scanned at a rate of 0.053 deg/s. Particle sizes of the Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were determined by measuring hydrodynamic diameter and particle size distribution in water using a Malvern Zetasizer Nano ZS90 (Malvern Panalytical Inc., MA, USA). A Cary 60 UV-Vis Spectrophotometer (Agilent Technologies, CA, USA) was used to determine absorption characterizations of particles. Excitation and emission spectra of Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were determined using FluoroMax-4 Spectrofluorometer, HORIBA Jobin Yvon. Gold, bismuth, and sulfur quantifications were performed by ICP-MS at the University of Nebraska-Lincoln's Spectroscopy and Biophysics Core Facility, via using an Agilent 7500cx ICP-MS (Santa Clara, CA, USA) coupled with a 96-well plate autosampler Model SC/DX4 from Elemental Scientific, Inc., operating in Mix-Gas collision/reaction mode (3.5 mL H 2 and 1.5 mL He per minute). Other conditions were plasma power, 1500 W; carrier gas flow, 1 L/minute; makeup gas flow, 0.15 L/minute; sample depth, 8 mm; plasma gas, 15 L/minute. The concentrations were calculated against an external calibration curve with 50 µg/L of Ga used as the internal standard (IS) throughout (Gallium-71 isotope). Tissue samples (liver, spleen, lung, kidney, Intestine and pancreas) were suspended in 4 times the volume of analytical grade nitric acid, incubated at room temperature for up to 2 hours, followed by overnight digestion at 65 ° C. The samples were cooled and diluted 20-fold into the autosampler to reach a 10 mg/mL final concentration. 2.4. Cell toxicity and uptake test UC-MSCs were cultured in MesenCult™ media supplemented with 2.5% v/v human platelet lysate and 1% v/v penicillin-streptomycin (P/S). The CellTiter-Glo™ assay was used to assess the cytotoxicity of the particles. Briefly, UC-MSCs were seeded at 10,000 cells per well in a 96-well clear bottom plate in the culture medium detailed above. Upon reaching 90% confluence, the cells were washed with PBS, serum-starved in MesenCult™ media without platelet lysate for 4 h, and subsequently replaced with fresh MesenCult™ media containing different concentrations of lipid-coated Au@Bi 2 S 3 NRs (0.5–200 µg/mL). After incubation for 12 h, the CellTiter-Glo™ assay was performed as per the manufacturer’s instructions. Untreated cells were used as controls. Fluorescence was measured using an IVIS Lumina II (Caliper Life Sciences) reader. Data was represented as fold change relative to the control. For determining the cellular uptake of the Au@Bi 2 S 3 NRs, the UC-MSCs were seeded at 1.5 × 10 6 cells per well in 12-well clear-bottom plates and cultured to 90% confluency in MesenCult™ media containing 2.5% human platelet lysate and 1% P/S. Subsequently, media containing FITC-Au@Bi 2 S 3 NRs at a concentration of 10 µg/mL was added to each well and incubated for 2 h to 12 h. Following incubation, the cells were washed, fixed with 4% v/v paraformaldehyde (PFA), and the nuclei were stained with DAPI. The uptake of fluorescent particles was assessed using Celigo image cytometer (model number 200-BFFL-5C; Nexcelom Bioscience LLC, CA, USA). To visualize the localization of particles within the cells, confocal microscopy was used. Briefly, cells were seeded on a pre-inserted coverslip in cell culture plate wells. Following the attachment of cells, the particles were added at a concentration of 10 µg/mL. After incubation for 12 h, the cells were fixed in 4% PFA, the nuclei were stained with DAPI, and imaged using a confocal Leica DMi8 Inverted Microscope (Leica Microsystems Inc., Illinois, USA). 2.5. CT Imaging of particles To assess Au@Bi 2 S 3 NRs at relevant CT imaging levels with Bi 2 S 3 NRs, Au@Bi 2 S 3 NRs were dispersed in DI water with different mass concentrations ranging from 11.4, 22.8 45.5, 91.2, to 182.3 µg/mL based on the bismuth concentration. ICP-MS analyses of lipid-coated Au@Bi 2 S 3 NRs suspension were shown with metal components: S = 283.48 µg/mL, Au = 440.11 µg/mL and Bi = 3,647.83 µg/mL concentration. Phantoms were scanned using a small animal scanner, Siemens Inveon PET/CT system (Siemens Medical Solutions, Knoxville, TN USA) with scanning parameters listed in the next section. 2.6. Biodistribution Rats (8–12 weeks old, male, Wistar) were purchased from Charles River Laboratories (USA) and housed according to Stanford University’s Administrative Panel for Laboratory Animal Care (APLAC). All procedures were performed in accordance with the regulations approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University. A total of 3 mL of lipid-coated FITC-Au@Bi 2 S 3 NRs with an ICP-MS quantitative S = 283.48 µg/mL, Au = 440.11 µg/mL, Bi = 3647.83 µg/mL concentration was injected intravenously (IV). Following the injection, the rats were anesthetized with isoflurane (3–4% for induction and 1–3% for maintenance) and imaged with CT after Au@Bi 2 S 3 NRs administration using the Siemens Inveon PET/CT system (Siemens Medical Solutions, Knoxville TN USA). CT settings were a 121 projection and SB70 mm installed pallet. X-ray source conditions were applied for 79 keV tube voltage, 493 µA of tube current, 50 µM of spot size, 0.5 mm of filter, 18.37 X 12.2 cm of FOV, 2.45 of scale, 482.26-150.17, Hot metal of scale bar. Inveon Acquisitions workplace viewer 4.0 software was used to acquire data. All animals were euthanized 120 hours post-injection of the Au@Bi2S3 NRs, using carbon dioxide as the method of euthanasia. 2.7. Histological analysis Histological analysis of the explanted organs (liver, spleen, pancreas, lung, and kidneys) at 120h following intravenous injection of Au@Bi 2 S 3 NRs was performed to assess the biocompatibility of our particles in small animals. The excised organs were fixed in 10% formalin, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E). Furthermore, immunohistochemistry using CD45 staining to assess any inflammatory response to Au@Bi 2 S 3 NRs was performed, as previously described [ 40 ]. The stained sections were analyzed using a NanoZoomer slide scanner 2.0-RS (Hamamatsu Photonics, Japan). 3. Results and Discussion 3.1 Au@Bi 2 S 3 NRs synthesis and characterization Solvothermal synthesis was used to synthesize Bi 2 S 3 NRs by mixing bismuth and sulfur precursors, such as thioacetamide and bismuth neodecanoate, dissolved in an organic solvent. The resulting solution was then placed in a high-pressure reactor autoclave and heated to the desired reaction temperature. As the bismuth and sulfur precursors react, they form bismuth sulfide, which precipitates as Bi 2 S 3 NRs. The resulting mixture was then cooled, and Bi 2 S 3 NRs were collected by centrifugation. Next, gold nanoparticles (AuNPs) were formed in situ on the surface of Bi 2 S 3 NRs by reducing HAuCl 4 3H 2 O with NaBH 4 to create Au@Bi 2 S 3 NRs. Here, gold ions, such as Gold (III) chloride trihydrate, were reduced onto the surface of Bi 2 S 3 NRs using sodium borohydride. Biocompatible hydrophilic NPs were then developed with the help of a uniform lipid coating (DSPE-PEG 2000 and phosphatidylcholine) using a film hydration method (Schematic 1) ; this reaction is carried out in an aqueous solution with the final Au@Bi 2 S 3 NRs purified for characterization using high-resolution transmission electron microscopy (HR-TEM), scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), X-ray fluorescence (XRF), and inductively coupled plasma mass spectroscopy (ICP-MS). Schematic for the synthesis of Au@Bi 2 S 3 NRs. Multimodal Au@Bi 2 S 3 NRs for high contrast X-ray CT imaging were obtained by core-shell structural synthesis. (a) Bi 2 S 3 NR cores were synthesized using a solvothermal synthetic route. (b) Bi 2 S 3 NRs were stabilized with a lipid coating, consisting of phosphatidylcholine and DSPE-PEG 2000 before then being coated with AuNPs on its surface to create Au@Bi 2 S 3 NRs. 3.1.1 HR-TEM, SAED and STEM HR-TEM was used to examine the crystal structure and lattice defects of Au@Bi 2 S 3 NRs, as well as their size, shape, and distribution. Low-power TEM images demonstrated that Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were of a uniform population having a rod shape of ~ 10 nm width and ~ 33–43 nm length (N = 20) ( Fig. 1 a-c ) . HR-TEM images showed AuNPs with a uniform spherical size (~ 3–5 nm) that were homogeneously distributed and attached to the surface of Bi 2 S 3 NRs ( Fig. 1 d-e ) . Furthermore, HR-TEM data also showed a lattice pattern with atoms at interplanar distances of 0.414 nm, 0.480 nm, 0.333 nm, and 0.260 nm, corresponding to the (220), (210), (130), and (311) planes, respectively (JCPDS 01-089-8963), which are in accordance with the atomic configuration of the Au@Bi 2 S 3 NRs. The SAED patterns of Au@Bi 2 S 3 NRs are shown by simulation indexation. The simulation index lines correspond to the rod's interplanar spacing ( Fig. 1 f ) [ 4 ]. SAED confirmed that the AuNPs were coordinated with the bismuth template on the surface of the Bi 2 S 3 NRs, which was assigned to the (111), (200), (220), (311), and (222) plane of gold (JCPDS; 04-007-4652; 03-065-3093 and 01-071-4073) for a face-centered cubic gold structure. The simulation indexation of the orthorhombic Bi 2 S 3 SAED pattern shows planes of (020), (211), (040), (131), (311), (231), (331), (060), (610), and (630) (JCPDS; 01-089-8963; 04-019-2866) [ 4 , 27 ], all of which confirm that Au@Bi 2 S 3 NRs were efficiently synthesized. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) illustrated the heterostructure and distribution of elements of bismuth (red), sulfur (blue), and gold (yellow) particles with mapping showing that the core of the particles was composed of Bi and S, while the shell was composed of Au (Fig. 1 g-l). 3.1.2 Structural characterization: EDX and XRD Energy dispersive X-ray spectroscopy (EDX) can identify the elemental composition of NPs by measuring the energy and intensity of X-rays emitted from the sample when it is bombarded with high-energy electrons; this provides detailed information about the structure and elemental composition of nanomaterials. EDX spectroscopy analysis demonstrated Bi, S, and Au signals with signature energy peaks detected in Au@Bi 2 S 3 NRs (Fig. 2a). It is important to note that EDX spectroscopy is a surface analysis technique that only provides information about the top few atomic layers of Au@Bi 2 S 3 NRs. Hence, we also used XRD to obtain more detailed information about the crystal structure of Au@Bi 2 S 3 NRs. Specifically, the XRD diffraction pattern was used to identify the presence of specific phases in the Au@Bi 2 S 3 NRs and to determine the crystal structure of those phases. The crystal structure and lattice planes of Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs were assessed using powder XRD studies. The XRD pattern of plain (undecorated) Bi 2 S 3 NRs showed signature peaks at 23.16° (220), 24.29° (130), 25.71° (310), 28.11° (021), 29.19° (211), 32.39° (221), 33.50° (301), 34.50° (311), 36.40° (240), 38.63° (231), 40.77° (141), 43.30° (421), 46.10° (002), 47.16° (431), 53.29° (222), 54.9° (351), 55.3° (061), 55.42° (360), and 59.23° (242), corresponding to a d-spacing of 0.396 nm, 0.356 nm, 0.352 nm, 0.324 nm, 0.310 nm, 0.280 nm, 0.271 nm, 0.263 nm, 0.251 nm, 0.245 nm, 0.225 nm, 0.211 nm, 0.198 nm, 0.194 nm, 0.177 nm, 0.173 nm, 0.167 nm and 0.155 nm, respectively, all of which belong to the orthorhombic structure (JCPDS: 01-089-8963) of Bi 2 S 3 NRs. For Au@Bi 2 S 3 NRs, along with the Bi 2 S 3 NRs peaks as mentioned above, additional peaks at 25.26° (110), 36.40° (111) and 43.30° (200), were observed for the planes that correspond to 0.355 nm, 0.235 nm, and 0.203 nm, respectively (JCPDS: 04-007-4652; 01-071-4073). XRD patterns with no anonymous peaks indicated that the synthesized nanomaterials were free from contaminants (Fig. 2b) . It is important to note that XRD is a non-destructive technique and does not provide information about the chemical composition of Au@Bi 2 S 3 NRs. To determine the chemical composition of Au@Bi 2 S 3 NRs, we used other techniques such as XPS, XRF spectrometer, and ICP-MS. 3.1.3 Chemical characterization: XPS, XRF, and ICP-MS. XPS is a surface-sensitive analytical technique that measures the elemental composition and chemical states of the elements present at the surface of Au@Bi 2 S 3 NRs (Fig. 2c-e). The survey spectrum revealed the presence of elements, such as Bi, S, O, and C, indicating the high purity of the particles. The peak for O arises, presumably, from adsorbed gases and/or oxides of C on the surfaces of the samples [ 28 ]; this observation is common in the case of ultrafine powder samples when exposed to atmospheric conditions. In XPS analysis of Au@Bi 2 S 3 NRs, the main peaks observed are due to the Bi, S, and S2p orbitals and Bi 4f peaks. High-resolution XPS spectra of Au 4f and Bi 4f (Figs. 2d and 2e) further elucidated the composition of the Au@Bi 2 S 3 NRs. For each asymmetric Bi 4f7/2 or Bi 4f5/2, the peak in both Bi 2 S 3 and Au@Bi 2 S 3 NRs could be deconvoluted into two peaks in the high-resolution XPS spectra in the Bi region (Fig. 2d). Peaks at 157.78 and 163.08 eV, which can be assigned to the binding energies of Bi 4f5/2 and Bi 4f7/2 in Bi 2 S 3 NRs, respectively, belong to lattice Bi bound to S (Bi-S bond). However, in Au@Bi 2 S 3 NRs, both peaks had shifted toward a lower intensity, meaning some Au atoms in Au@Bi 2 S 3 NRs may bind to S atoms to form an Au-S bond, which is in line with thiols demonstrating a high affinity for Au. The high-resolution XPS spectra in the Au region (Fig. 2e) showed two peaks at 85.88 and 89.68 eV, which can be assigned to the binding energies of Au 4f5/2 and Au 4f7/2 in Au@Bi 2 S 3 , respectively, thereby indicating the presence of Au in the Au@Bi 2 S 3 NRs nanostructures. The broad peak at around 224.68 eV corresponds to the binding energy of S2s ( Figure S1 a ). Figure S1 b shows the peaks of S 2p3/2 and S 2p1/2 located at 165.58 and 160.28 eV, respectively, which match the literature values of the sulfide anion (S 2− ) ( Figure S1 b ). The observed values were found to be in close agreement with data reported by Grigas et al [ 29 ]. Collectively, the data showed that Au@Bi 2 S 3 NRs have a uniform composition with bimetallic Bi/S cores and Au shells. Overall, XPS is a powerful tool for studying the surface chemistry of Au@Bi 2 S 3 NRs and provides valuable information about the elemental composition of Bi 2 S 3 . In addition, the mass distribution of Au, Bi, and S was determined using XRF analysis. The Bi 2 S 3 and Au@Bi 2 S 3 NRs exhibited Bi/S = 83.49/15.84 mass percent (%) and Bi/S/Au = 74.53/16.69/8.77 mass percent (%), respectively ( Figure S2 ). Moreover, Bi/Au ratio = 8.28:1% w/w content in lipid-decorated Au@Bi 2 S 3 NRs was determined by ICP-MS. These results indicate that the ratio of Bi/Au by ICP-MS and XRF matched. 3.1.4 Particle size and zeta potential analysis Dynamic light scattering (DLS) was used to evaluate the average size, polydispersity, and zeta potential analysis of particles (Fig. 2f) . The particles size of Au@Bi 2 S 3 NRs and Bi 2 S 3 NRs were 224.6 ± 2.4 nm and 206.6 ± 8.3nm, respectively. The particle size of Au@Bi 2 S 3 NRs was ~ 20 nm bigger than the size of Bi 2 S 3 NRs because of the AuNPs decoration. The polydispersity index, which measures the sample's heterogeneity based on size, was comparable between the particles (0.2 and 0.3 for Bi 2 S 3 NRs and Au@Bi 2 S 3 NRs, respectively). Moreover, the DLS showed an average zeta potential of -26.46 ± 2.43 mV for the Bi 2 S 3 NRs and − 45.76 ± 1.02 mV for Au@Bi 2 S 3 NRs, which demonstrates an increase of ~-20 mV for Au@B 2 S 3 NRs compared to B 2 S 3 NRs. The higher negative surface charge of Au@Bi 2 S 3 NRs was due to gold particles, which confer particle stability preventing any aggregation in aqueous solution. It is important to note that particle size and zeta potential are two important characteristics that can significantly affect biodistribution and stability [ 30 – 32 ]. Figure 2. Structural and surface chemistry characterization of particles. (a) Energy-dispersive X-ray (EDX) elemental mapping of Bi, S, and Au, with Cu originating from the TEM grid. (b) X-ray diffraction (XRD) patterns of Bi₂S₃ NRs and Au@Bi₂S₃ NRs with indexed peaks corresponding to expected crystallographic planes. (c–e) X-ray photoelectron spectroscopy (XPS) analysis: (c) full survey spectrum, (d) Bi region, and (e) Au region of Au@Bi₂S₃ NRs. (f) Hydrodynamic size distribution of Bi₂S₃ NRs and Au@Bi₂S₃ NRs measured by dynamic light scattering (DLS). 3.2. Cellular uptake and toxicity evaluation To evaluate the biocompatibility of Au@Bi 2 S 3 NRs, we tested their cell uptake and toxicity using umbilical cord derived mesenchymal stem cells (UC-MSCs), given the potential to use this nanoparticle to label and track these cells. Fluorescence-based CellTiter-Glo™ assays were used to test UC-MSC viability after exposure for 12 h to different concentrations of Au@Bi 2 S 3 NRs (0.5 to 200 µg/mL). Viable cells showed fluorescence, while dead cells have a loss of fluorescence. It was noted that cell viability remained about 100% between 0.5–40 µg/mL, but this drastically decreased at higher concentrations (60–200 µg/mL) (Figure S3) . Next, the effect of cell uptake of FITC-Au@Bi 2 S 3 NRs was studied. Representative confocal images of FITC-Au@Bi 2 S 3 NRs (10 µg/mL) incubated with UC-MSCs for 12 h are reported in Figure S3c , which shows green fluorescence in the cell cytoplasm (FITC staining of the particles) and blue nucleus (DAPI staining of cell nuclei). To further verify in vitro uptake of Au@Bi 2 S 3 NRs in UC-MSCs, FITC-Au@Bi 2 S 3 NRs cellular uptake and intracellular localization of particles within UC-MSCs were assessed using a Celigo Imaging Cytometer in a time-series dynamic study (up to 12 h). Cellular uptake of FITC-Au@Bi 2 S 3 NRs was observed to occur in a time-dependent manner. Images show an increased amount of particle aggregates inside the cytoplasm of UC-MSCs over time (after 2h through 12h), as noted by the increased number of black particles or yellow dots in the bright-field and fluorescence images, respectively (Figure S3d) . Particle uptake was relatively slow during the first few hours of incubation before reaching saturation at 10–12 hours (Figure S3d). It is important to note that the uptake of Au@Bi 2 S 3 NRs by UC-MSCs, as well as their potential effects on UC-MSCs, can vary depending on the specific study and the conditions used. Previous studies show that various factors such as the size, surface properties, concentration, and exposure time affect the particle uptake in cells and tissue [ 4 ], and their cellular uptake can occur through several mechanisms, including endocytosis, phagocytosis, and pinocytosis [ 1 , 4 , 33 ]. 3.3. In vivo biodistribution and clearance The feasibility of utilizing Au@Bi 2 S 3 NRs as a contrast agent for visualizing soft tissues was assessed through CT imaging (Fig. 3 a). Initially, we compared the contrast efficacy of Au@Bi 2 S 3 NRs with that of mesoporous silica nanoparticles (SiNPs) in dry powder form (Fig. 3 b). Our results demonstrated that the combination of bismuth and gold in Au@Bi 2 S 3 NRs produced significantly higher contrast levels compared to both Bi 2 S 3 NRs and SiNPs. (Fig. 3 b-c). Next, we investigated the in vivo biodistribution of Au@Bi 2 S 3 NRs following intravenous injection in rats, utilizing whole-body CT imaging with static scans conducted at various time points: 24, 48, 72, 96, and 120 hours post-injection. The data presented here focuses exclusively on the 24-hour time point, excluding information from the other time intervals. The 24-hour post-injection scan (Fig. 3 d) revealed that Au@Bi 2 S 3 NRs predominantly localized in major organs, including the liver, spleen, lung, and pancreas. This finding was further validated through ex vivo imaging of the harvested organs (Fig. 3 d), which were collected at 120 hours after particle injection. Notably, the signal intensity at 120 hours in the liver and spleen was significantly stronger compared to that in the lung and pancreas, a trend corroborated by ICP-MS measurements. Additionally, ICP-MS results indicated that the bismuth content in various organs aligned with the signal intensity observed in CT imaging at the 120-hour mark. Accumulation of Au@Bi 2 S 3 NRs was confirmed in organs, with bismuth concentrations measured at 893.1 ± 107.7 µg/g in the spleen, 714.4 ± 118.1 µg/g in the liver, 126.4 ± 22.5 µg/g in the lung, 7.8 ± 1.1 µg/g in the pancreas, 74.9 ± 4.7 µg/g in the kidney, and 4.0 ± 0.7 µg/g in the intestine ( Figure S4 ). Hence, the extended imaging window following intravenous administration of Au@Bi 2 S 3 NRs presents significant potential for various clinical applications. The biodistribution and subsequent biological effects of NPs depend on a variety of factors, including their size, shape, core materials, and method of administration, to name a few. Following IV administration, Au@Bi 2 S 3 NRs, are predominantly taken up by the liver and spleen, as also shown by several other studies that have examined Bi 2 S 3 NRs [ 4 , 34 , 35 ]; this is in keeping with the function of organs of the reticuloendothelial system, of which the liver and spleen are part of, which remove and eliminate foreign substances from the bloodstream. Once in these organs, NPs are then processed. Here, there will be a balance between NPs clearance and potential ongoing inflammation (due to how the body processes the particles) that could result in potential local cellular injury [ 4 , 36 – 38 ]. 3.4 Histological assessments The liver, spleen, lung, kidney, and pancreas demonstrated normal gross size and morphology at the end of our study. Subjectively, Au@Bi 2 S 3 NRs increased the darkness of the color of the liver and spleen compared to control animals ( Fig. 4 a ) with H&E-stained samples (Fig. 4 b) showing the particles evenly distributed throughout the parenchyma of each organ with no surrounding inflammatory cell infiltrate at 5 days. Moreover, fewer Au@Bi 2 S 3 NRs were seen in the lungs, pancreas, and kidney samples compared to the liver and spleen. Immunohistochemical staining, using CD45 (Fig. 4 c), also indicated no leukocyte infiltration. Collectively, Au@Bi 2 S 3 NRs elicited minimal-to-no inflammatory response in the short term. Future studies will aim to evaluate how these organs will handle Au@Bi 2 S 3 NRs to determine their long-term safety profile. 4. Conclusion In this work, we presented a simple technique to synthesize Au@Bi 2 S 3 NRs via a solvothermal method and state-of-the-art characterization of Au@Bi 2 S 3 NRs. By using a lipid-based phase changing agent (DSPE-PEG 2000) as a template for the functionalization of AuNPs onto Bi 2 S 3 NRs, instead of Tween 20 that has been previously reported [ 39 ], we have been able to control and optimize particle size, crystallinity, and reproducibility of Au@Bi 2 S 3 NRs. Au@Bi 2 S 3 NRs were also seen to enhance soft tissue contrast on CT imaging of living animals and, hence, are promising nanoparticles for further development for both multimodal imaging as well as theranostic applications. Declarations Acknowledgments The authors thank Histo-tech Laboratory Inc. for histology slide preparation and H&E staining. Author Contributions B.K. conceptualized, prepared, and characterized the nanorods; performed the in vivo experiments; conducted data collection and analysis and led manuscript preparation and writing. R.P. assisted with nanorod characterization, in vivo experiments, histology, data analysis, and manuscript preparation and editing. G.S. performed the cell uptake and toxicity experiments and data analysis. J.Z. edited and proofread the manuscript. A.S. acquired high-resolution transmission electron microscopy images and assisted with image analysis. R. Prasad draft overview and editing. B.D. edited and proofread the manuscript. J.W. performed the animal experiments. K.H. edited and proofread the manuscript. P.D. provided expert revisions. A.S.T. conceptualized and designed the experiments, supervised the project, and contributed to manuscript preparation and writing. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by funding from the Department of Radiology at Stanford University, research grants from the NIDDK (R01DK129598, R01DK129343, R01DK141802), and the Akiko Yamazaki and Jerry Yang Faculty Scholarship. Data Availability Statement The authors confirm that the data supporting the findings of this study are available within this article and its Supplementary material. Raw data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics Approval and Consent to Participate. The authors declare that all experiments in the present study were approved by the Administrative Panel on Laboratory Animal Care at Stanford University and were carried out per the Institutional Animal Care and Use Committee (IACUC) approval (Title: In vivo studies of engineered three-dimensional porous bio-scaffolds for pancreaticislet transplantation; Approval number: 32821; Last Approval date: 10/15/2024). Consent to Publish. Not applicable. Competing Interests. The authors declare no competing interest References Augustine R, Hasan A, Primavera R, Wilson RJ, Thakor AS, Kevadiya BD (2020) Mater Today Commun 25:101692. 10.1016/j.mtcomm.2020.101692 Kim D, Kim J, Park YI, Lee N, Hyeon T (2018) ACS Cent Sci 4(3):324–336. 10.1021/acscentsci.7b00574 Sukhanova A, Bozrova S, Sokolov P, Berestovoy M, Karaulov A, Nabiev I (2018) Nanoscale Res Lett 13(1):44. 10.1186/s11671-018-2457-x Kevadiya BD, Ottemann B, Mukadam IZ, Castellanos L, Sikora K, Hilaire JR, Machhi J, Herskovitz J, Soni D, Hasan M, Zhang W, Anandakumar S, Garrison J, McMillan J, Edagwa B, Mosley RL, Vachet RW, Gendelman HE (2020) Theranostics 10 (2), 630–656. 10.7150/thno.39847 Patel PR, De Jesus O (2022) CT Scan. In StatPearls, StatPearls Publishing Copyright © 2022, StatPearls Publishing LLC. Treasure Island (FL) Miwa S, Otsuka T, Orthop Sci J (2017) 22 (3), 391–400. 10.1016/j.jos.2017.01.006 Patel M, Tann M, Liangpunsakul S (2021) Am J Med Sci 362(3):252–259. 10.1016/j.amjms.2020.10.031 PAUWELS E, VAN LOO D, CORNILLIE P, BRABANT L, VAN HOOREBEKE L (2013) J Microsc 250(1):21–31. https://doi.org/10.1111/jmi.12013 Yeh BM, FitzGerald PF, Edic PM, Lambert JW, Colborn RE, Marino ME, Evans PM, Roberts JC, Wang ZJ, Wong MJ, Bonitatibus PJ (2017) Jr Adv Drug Deliv Rev 113:201–222. 10.1016/j.addr.2016.09.001 Rabin O, Manuel Perez J, Grimm J, Wojtkiewicz G, Weissleder R (2006) Nat Mater 5(2):118–122. 10.1038/nmat1571 Andreucci M, Solomon R, Tasanarong A (2014) BioMed research international 2014, 741018–741018. 10.1155/2014/741018 Webb JA, Stacul F, Thomsen HS, Morcos SK (2003) Members of the *Contrast Media Safety Committee of the European Society of Urogenital Radiology. Eur Radiol 13(1):181–184. 10.1007/s00330-002-1650-5 Singh J, Daftary A (2008) J Nucl Med Technol 36(2):69–74. 10.2967/jnmt.107.047621 He F, Ji H, Feng L, Wang Z, Sun Q, Zhong C, Yang D, Gai S, Yang P, Lin J (2021) Biomaterials 264, 120453. 10.1016/j.biomaterials.2020.120453 Mahan MM, Doiron AL (2018) Journal of Nanomaterials 2018, 5837276. 10.1155/2018/5837276 Subramanyam P, Vinodkumar T, Deepa M, Subrahmanyam C (2019) J Mater Chem C 7(21):6398–6405. 10.1039/C9TC00759H Herskovitz J, Hasan M, Machhi J, Mukadam I, Ottemann BM, Hilaire JR, Woldstad C, McMillan J, Liu Y, Seravalli J, Sarella A, Gendelman HE, Kevadiya BD (2021) Nanotheranostics 5 (4), 417–430. 10.7150/ntno.59568 Kevadiya BD, Woldstad C, Ottemann BM, Dash P, Sajja BR, Lamberty B, Morsey B, Kocher T, Dutta R, Bade AN, Liu Y, Callen SE, Fox HS, Byrareddy SN, McMillan JM, Bronich TK, Edagwa BJ, Boska MD, Gendelman HE (2018) Theranostics 8 (1), 256–276. 10.7150/thno.22764 Elahi N, Kamali M, Baghersad MH (2018) Talanta 184, 537–556. 10.1016/j.talanta.2018.02.088 Cabuzu D, Cirja A, Puiu R, Grumezescu AM (2015) Curr Top Med Chem 15(16):1605–1613. 10.2174/1568026615666150414144750 Sonavane G, Tomoda K, Makino K, Colloids, Surfaces B, Biointerfaces (2008) 66 (2), 274–280. 10.1016/j.colsurfb.2008.07.004 Ross RD, Cole LE, Tilley JMR, Roeder RK (2014) Chem Mater 26(2):1187–1194. 10.1021/cm4035616 Li L, Lu Y, Jiang C, Zhu Y, Yang X, Hu X, Lin Z, Zhang Y, Peng M, Xia H, Mao C (2018) Adv Funct Mater 28(5). 10.1002/adfm.201704623 Ai K, Liu Y, Liu J, Yuan Q, He Y, Lu L (2011) Adv Mater 23(42):4886–4891. 10.1002/adma.201103289 Liu J, Zheng X, Yan L, Zhou L, Tian G, Yin W, Wang L, Liu Y, Hu Z, Gu Z, Chen C, Zhao Y (2015) ACS Nano 9(1):696–707. 10.1021/nn506137n Zheng X, Shi J, Bu Y, Tian G, Zhang X, Yin W, Gao B, Yang Z, Hu Z, Liu X, Yan L, Gu Z, Zhao Y (2015) Nanoscale 7(29):12581–12591. 10.1039/c5nr03068d Zheng X, Shi J, Bu Y, Tian G, Zhang X, Yin W, Gao B, Yang Z, Hu Z, Liu X, Yan L, Gu Z, Zhao Y (2015) Nanoscale 7(29):12581–12591. 10.1039/c5nr03068d Wang H, Zhu J-J, Zhu J-M, Chen H-Y (2002) J Phys Chem B 106(15):3848–3854. 10.1021/jp0135003 Grigas J, Talik E, Lazauskas V (2002) physica status solidi (b) 232 (2), 220–230. 10.1002/1521-3951(200208)232:2%3C220::AID-PSSB220%3E3.0.CO;2-F Sonavane G, Tomoda K, Makino K, Colloids Surf BB (2008) 66 (2), 274–280. 10.1016/j.colsurfb.2008.07.004 Dreaden EC, Austin LA, Mackey MA, El-Sayed MA (2012) Ther Deliv 3(4):457–478. 10.4155/tde.12.21 Hoshyar N, Gray S, Han H, Bao G (2016) 673 – 92. Nanomed (Lond) 11(6). 10.2217/nnm.16.5 Behzadi S, Serpooshan V, Tao W, Hamaly MA, Alkawareek MY, Dreaden EC, Brown D, Alkilany AM, Farokhzad OC, Mahmoudi M (2017) Chem Soc Rev 46(14):4218–4244. 10.1039/c6cs00636a Badrigilan S, Heydarpanahi F, Choupani J, Jaymand M, Samadian H, Hoseini-Ghahfarokhi M, Webster TJ, Tayebi L (2020) Int J Nanomed 15:7079–7096. 10.2147/ijn.S250001 Yu M, Zheng J, Nano ACS (2015) 9 (7), 6655–6674. 10.1021/acsnano.5b01320 Nel A, Xia T, Mädler L, Li N (2006) Science 311(5761):622–627. 10.1126/science.1114397 Dobrovolskaia MA, McNeil SE (2007) Nat Nanotechnol 2(8):469–478. 10.1038/nnano.2007.223 Gustafson HH, Holt-Casper D, Grainger DW, Ghandehari H (2015) Nano Today 10(4):487–510. 10.1016/j.nantod.2015.06.006 Cheng Y, Chang Y, Feng Y, Jian H, Tang Z, Zhang H (2018) Angew Chem Int Ed 57(1):246–251. 10.1002/anie.201710399 Primavera R, Razavi M, Kevadiya BD, Wang J, Vykunta A, Di Mascolo D, Decuzzi P, Thakor AS (2021) Biofabrication 13 (3), 035011. 10.1088/1758-5090/abdcac Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8854864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":603196384,"identity":"c14846c5-643b-4c4e-8c31-2f8b60080b57","order_by":0,"name":"Bhavesh D Kevadiya","email":"","orcid":"","institution":"Stanford University, School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bhavesh","middleName":"D","lastName":"Kevadiya","suffix":""},{"id":603196385,"identity":"42aea4b6-546e-4d5b-8302-e4fe69c92d5a","order_by":1,"name":"Rosita Primavera","email":"","orcid":"","institution":"Stanford University, School of 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18:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8854864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8854864/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104548354,"identity":"10666c01-4df0-4fb9-b459-cf4538093989","added_by":"auto","created_at":"2026-03-13 07:41:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":371113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of Au@Bi\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e NRs.\u003c/strong\u003e Low-resolution TEM images show \u003cstrong\u003e(a)\u003c/strong\u003e Bi₂S₃ nanorods (NRs) and \u003cstrong\u003e(b)\u003c/strong\u003e AuNP-decorated Bi₂S₃ NRs (Au@Bi₂S₃ NRs). \u003cstrong\u003e(c)\u003c/strong\u003e Magnified TEM image displaying AuNPs distributed uniformly across the Bi₂S₃ NR surface. \u003cstrong\u003e(d–e)\u003c/strong\u003e HR-TEM images of Au@Bi₂S₃ NRs showing surface Au and the Bi₂S₃ core; the inset displays lattice fringes with measured spacings of 0.414 nm, 0.333 nm, 0.260 nm, and 0.480 nm, corresponding to Bi₂S₃ crystallographic planes (220, 130, 311, and 210). \u003cstrong\u003e(f)\u003c/strong\u003e Selected-area electron diffraction (SAED) pattern of Au@Bi₂S₃ NRs with indexed diffraction rings consistent with expected interplanar spacings. \u003cstrong\u003e(g) \u003c/strong\u003eHAADF-STEM image of Au@Bi₂S₃ NRs. Elemental STEM maps show the distribution of \u003cstrong\u003e(h)\u003c/strong\u003e Bi, \u003cstrong\u003e(i)\u003c/strong\u003e S, and \u003cstrong\u003e(j)\u003c/strong\u003e Au. \u003cstrong\u003e(k–l)\u003c/strong\u003e Overlay maps combining Bi, S, and Au elements alongside the corresponding HAADF signal.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/cb47d2e99cb1a57d4d2ad030.png"},{"id":104548189,"identity":"ed1a9088-b949-4b77-bf08-bfb7fdc708a9","added_by":"auto","created_at":"2026-03-13 07:41:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and surface chemistry characterization of particles.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(a)\u003c/strong\u003e Energy-dispersive X-ray (EDX) elemental mapping of Bi, S, and Au, with Cu originating from the TEM grid. \u003cstrong\u003e(b)\u003c/strong\u003e X-ray diffraction (XRD) patterns of Bi₂S₃ NRs and Au@Bi₂S₃ NRs with indexed peaks corresponding to expected crystallographic planes. \u003cstrong\u003e(c–e)\u003c/strong\u003e X-ray photoelectron spectroscopy (XPS) analysis: \u003cstrong\u003e(c)\u003c/strong\u003efull survey spectrum, \u003cstrong\u003e(d)\u003c/strong\u003e Bi region, and \u003cstrong\u003e(e)\u003c/strong\u003e Au region of Au@Bi₂S₃ NRs. \u003cstrong\u003e(f)\u003c/strong\u003e Hydrodynamic size distribution of Bi₂S₃ NRs and Au@Bi₂S₃ NRs measured by dynamic light scattering (DLS).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/6f65cef40a1250240e913425.png"},{"id":104548486,"identity":"bf20f545-7b3c-4ee1-a3f1-6ef5c95d2b9f","added_by":"auto","created_at":"2026-03-13 07:42:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":508845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo biodistribution of Au@Bi₂S₃ NRs. (a)\u003c/strong\u003e Experimental timeline for CT imaging studies in Wistar rats following intravenous administration of Au@Bi₂S₃ NRs. \u003cstrong\u003e(b)\u003c/strong\u003eCT images of Au@Bi₂S₃ NRs compared with silica nanoparticles (SiNPs) powder and water controls. \u003cstrong\u003e(c)\u003c/strong\u003e CT phantom images of Bi₂S₃ NRs and Au@Bi₂S₃ NRs prepared at concentrations of 11.4, 22.8, 45.6, 91.2, and 182.4 µg/mL (left-to-right) in PBS. \u003cstrong\u003e(d)\u003c/strong\u003e Coronal-plane in vivo CT images acquired 120 h after IV injection of Au@Bi₂S₃ NRs. Corresponding whole body and ex vivo CT images of harvested liver, spleen, lung, and pancreas collected at 120 h.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/09d1ac0ae024f6fd594b6d8a.png"},{"id":104548586,"identity":"6adbbc23-dec7-4fcd-b7cb-28a64ea380aa","added_by":"auto","created_at":"2026-03-13 07:43:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":737969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological assessment. (a)\u003c/strong\u003e Representative photograph of excised organs collected 120 h after IV administration of Au@Bi₂S₃ NRs. \u003cstrong\u003e(b)\u003c/strong\u003e Hematoxylin and eosin (H\u0026amp;E) staining of liver, spleen, pancreas, lung, and kidney sections. \u003cstrong\u003e(c)\u003c/strong\u003e Immunohistochemical staining for CD45 using DAB (3,3′-diaminobenzidine) chromogen. Black punctate signal in H\u0026amp;E sections indicate intracellular particle deposits. CD45 staining highlights leukocyte presence within tissue sections.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/17df95333cc91248347d88e9.png"},{"id":104548677,"identity":"673a168e-0d20-4509-b809-2fa1c746d254","added_by":"auto","created_at":"2026-03-13 07:43:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2887029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/ecf7cc1d-a825-4e4e-895d-8535d1ee5eac.pdf"},{"id":104548451,"identity":"fc4048d6-df0d-4b90-ad26-8e4a58dc7b98","added_by":"auto","created_at":"2026-03-13 07:42:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":115019272,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFIGURE.docx","url":"https://assets-eu.researchsquare.com/files/rs-8854864/v1/63c4128ff467ce4abf577877.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDevelopment of Gold-Bismuth Sulfide Nanorods (Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs) for X-ray- based imaging\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMetal nanoparticles (NPs) have unique properties that make them ideal for bioimaging applications, but their potential toxicity must be carefully considered [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The high-energy blocking capabilities of metal NPs make them useful for computed tomography (CT) imaging [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CT is a widely employed imaging modality that utilizes X-rays to generate detailed three-dimensional images of the body, relying on the varying densities of tissues to block X-rays to different extents [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite its effectiveness, distinguishing between tissues with similar densities can be challenging due to subtle differences in X-ray absorption, which may complicate image interpretation. To address this issue, contrast agents are employed. These agents can either accumulate in specific tissues or fill luminal spaces, such as blood vessels or the gastrointestinal tract, thereby enhancing X-ray absorption and improving image contrast [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Commonly used agents, like iodine and barium, can increase the radiodensity of tissues within which they reside, and are generally considered safe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], though they can be associated with adverse reactions, thyroid gland dysfunction, and nephropathy [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, the transit of these conventional contrast agents through tissues is relatively rapid, especially when administered via a vascular route; accordingly, precise timing is required for imaging or repeated CT dosing. However, these issues can potentially be improved by using electron-dense nanomaterials that can be retained longer in tissues [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, metal NPs have become increasingly utilized as medical imaging agents due to their improved biodistribution, biostability, predictable cellular interactions, and reproducible synthesis, especially compared to traditional contrast agents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Indeed, gold (Au) and bismuth (Bi) based NPs have been well studied as potential contrast agents for CT imaging [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Spherical AuNPs have several advantages, including their high atomic number, high X-ray attenuation, non-toxicity, facile synthesis, and surface functionalization for colloidal stability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Rod-shaped bismuth sulfide nanorods (Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs) also have strong X-ray absorption, excellent biocompatibility, high stability, and relatively long circulation times [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. By combining the properties of both these NPs, hybrid bimetal NPs can be created, which may offer improved imaging performance compared to either type of metal NPs alone.\u003c/p\u003e \u003cp\u003eThe synthesis of both AuNPs and Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003eNRs is a challenging task, given their rapid oxidation, which decreases their stability and shelf life. Accordingly, these NPs require the use of hazardous chemicals and manufacturing processes with high production costs, which can limit their clinical translation. In the present study, we developed an approach to reproducibly make a hybrid metal gold-bismuth-sulfide particle (i.e. Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs) of high quality, uniformity, and stability using a solvothermal synthesis approach. Here, spherical AuNPs were decorated onto the surface of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs in situ, and then Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were bioanalytically characterized using state-of-the-art tools, followed by in vitro and in vivo biocompatibility testing and in vivo imaging using CT.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003eGold (III) chloride trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO), thioacetamide (CH\u003csub\u003e3\u003c/sub\u003eCSNH\u003csub\u003e2\u003c/sub\u003e), Bismuth neodecanoate (Bi(OCOC(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e5\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e), oleic acid (OA), oleylamine (OAm, 70%), and L-α-phosphatidylcholine (PC) (from egg yolk), were obtained from MilliporeSigma, St. Louis, MO, USA. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-fluorescein (sodium salt) (FITC-DSPE-PEG 2,000) was obtained from Xi'an ruixi Biological Technology Co, China. DSPE-PEG 2,000 (N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) was obtained from NOF Corporation, Tokyo, Japan. Human Wharton\u0026rsquo;s Jelly-derived mesenchymal stem cells (UC-MSCs) were obtained from StemBioSys, San Antonio, TX, USA. DAPI (4',6-diamidino-2-phenylindole) was obtained from Thermo Fisher Scientific, USA. Cell culture media MesenCult\u0026trade; and human platelet lysate were obtained from Stemcell Technologies, Vancouver, BC, CA. CellTiter-Glo\u0026trade; was purchased from Promega, Madison, WI, USA. Male rats were purchased from Charles River Laboratories, USA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs\u003c/h2\u003e \u003cp\u003eCore bismuth sulfide (Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e) nanorods (NRs) were synthesized using the solvothermal method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] followed by decoration with AuNPs. Briefly, 1.45 g (~\u0026thinsp;1.87 mmol) of bismuth neodecanoate and 20 mL of OA were placed into a 500 mL capacity Teflon-lined autoclave reactor and the mixture was stirred for 10 minutes. Subsequently, 10 mL of ethanol was added dropwise and mixed for 10 minutes at 300 rpm. In a separate glass vial, 150 mg of thioacetamide (~\u0026thinsp;1.99 mmol) was mixed with 4 mL of OAm and sonicated until a uniform, light yellow-colored solution was obtained. The thioacetamide-OAm mixture was then transferred to a Teflon-lined stainless-steel autoclave, and the solution turned black-colored. This mixture was stirred for 1 h, placed in a sealed autoclave that was maintained at 150\u0026deg;C for 8 h, and then allowed to cool to room temperature. The precipitate was collected by centrifugation, washed several times with 200-proof ethanol, and dried overnight in a desiccator. The Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were finally obtained as a black powder. The Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were decorated with AuNPs using a seed-mediated growth method, which involved the following steps: Step one - Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (25 mg) powder was dispersed in anhydrous chloroform (1 mL). DSPE-PEG 2000 (25 mg) was mixed in chloroform (1 mL) in a separate glass vial. Both components were bath sonicated to ensure they were completely dispersed and dissolved. Step two - the solutions were mixed with 2 mL of DI water, and the solvent was then evaporated slowly by heating at 80\u0026deg;C in a glass vial. Step three - the Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs obtained as a black homogenous mixture was dispersed in 50 mL of DI water and heated it at 80\u0026deg;C for 30 min while stirring at 500 rpm. Step four\u0026thinsp;\u0026minus;\u0026thinsp;1 mL of a HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO (10 mmol) aqueous solution was added to Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e-DSPE-PEG 2000 solution. After 20 min, a reducing agent, 600 \u0026micro;L of ice-cold NaBH\u003csub\u003e4\u003c/sub\u003e solution in DI water (1 mg/mL) was introduced to initiate the surface-confined decorating of uniform AuNPs while stirring for an additional 30 min at 95\u0026deg;C at 500 rpm. During this time, the colloidal solution changed in color from black to brownish-black, indicating the formation of AuNPs decorating Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003eNRs). Step five - Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were further purified by centrifugation at 7,000 rpm for 30 min, washed with DI water and dispersed in DI water for further characterization. The resultant AuNPs were typically\u0026thinsp;~\u0026thinsp;2\u0026ndash;3 nm in diameter, and these can be easily tuned by modulating the amount of gold ions used. For cell and animal testing, lipid-coated Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were prepared using a thin-film dispersion method. Briefly, DSPE-PEG 2000 and PC (50:50% w/w) were dissolved in 5 mL of chloroform in a round-bottom flask to afford a thin film upon solvent evaporation, which was subsequently vacuum dried. Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (~\u0026thinsp;20 mg), dispersed in cyclohexane and mixed with 1% (v/v) Tween-80, were sonicated. Cyclohexane was evaporated from the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs/cyclohexane/Tween-80 emulsion. A Tween-80-coated Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs solution was next added to the lipid film flask and dispersed in a lipid film at 45\u0026deg;C with bath sonication. The product was collected after centrifugation at 500 rpm for 10 minutes and then dispersed in MilliQ water for further characterization and CT imaging testing. FITC-DSPE-PEG 2000 sodium salt was used as a fluorescence conjugate. The FITC-DSPE-PEG 2000 mass ratios were selected as 0.25% w/w with PC and DSPE-PEG2000 to form a lipid film for FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and to use for cell study. The remaining purification steps of the procedure were the same as above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs\u003c/h2\u003e \u003cp\u003eThe morphology, lattice crystal structure, elemental composition, and chemical color mapping of the synthesized Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were determined by brightfield high-resolution transmission electron microscopy (HR-TEM), selected area electron diffraction (SAED), energy-dispersive X-ray (EDX) spectroscopy, and scanning transmission electron microscopy (STEM) with high-angle annular dark-field (HAADF) (FEI Tecnai Osiris S/TEM). The TEM samples were prepared in cyclohexane and dried on a copper grid (Electron Microscopy Sciences-EMS, Hatfield, PA, USA) at room temperature. The additional gain in speed can also be used to collect EDX elemental mappings from a larger field-of-view. The lattice fringes of the obtained samples and the corresponding SAED patterns were examined using HR-TEM at 200 kV. The experimental SAED patterns were analyzed using PCED2.0 software.\u003c/p\u003e \u003cp\u003eThe particles were also characterized by performing wavelength dispersive X-ray fluorescence (WDXRF) analysis using a Rigaku WDXRF (Supermini200) spectrometer with high resolution and lower detection limits for elemental analysis. A 200 W, air-cooled, Pd X-ray source was operated at 50 kV and 4 mA to produce excitation spectra with faster elemental detection capability. A three-crystal analyzing unit was equipped in the system to support the standard LiF (200). Nanoparticle surface chemistry analysis was performed with X-ray photoelectron spectroscopy (XPS) and measurements were carried out using monochromatic Al K-alpha (α) X-ray with an energy of 1486.6 eV by Thermo Scientific K-alpha\u0026thinsp;+\u0026thinsp;XPS (Thermo Fisher Scientific, Waltham, MA, USA). Powder X-ray diffraction (XRD) analyses of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were performed in the 2θ range of 2\u0026ndash;60\u0026deg; using a PANalytical Empyrean diffractometer (PANalytical Inc.; Westborough, MA, USA) with Cu-Kα radiation (1.5418 \u0026Aring;) at 40 kV, 45 mA settings. A mask of 20 mm and a divergence slit of 1/32\u0026deg; were used for the incident beam path. A thin layer of the nanoparticle powder sample was placed on a zero-background silicon plate and the sample holder then continuously spun at the rate of 22.5 deg/s during all measurements. The PIXcel3D detector, equipped with a beam monochromator (PANalytical Inc.; Westborough, MA, USA) was scanned at a rate of 0.053 deg/s.\u003c/p\u003e \u003cp\u003eParticle sizes of the Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were determined by measuring hydrodynamic diameter and particle size distribution in water using a Malvern Zetasizer Nano ZS90 (Malvern Panalytical Inc., MA, USA). A Cary 60 UV-Vis Spectrophotometer (Agilent Technologies, CA, USA) was used to determine absorption characterizations of particles.\u003c/p\u003e \u003cp\u003eExcitation and emission spectra of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were determined using FluoroMax-4 Spectrofluorometer, HORIBA Jobin Yvon. Gold, bismuth, and sulfur quantifications were performed by ICP-MS at the University of Nebraska-Lincoln's Spectroscopy and Biophysics Core Facility, via using an Agilent 7500cx ICP-MS (Santa Clara, CA, USA) coupled with a 96-well plate autosampler Model SC/DX4 from Elemental Scientific, Inc., operating in Mix-Gas collision/reaction mode (3.5 mL H\u003csub\u003e2\u003c/sub\u003e and 1.5 mL He per minute). Other conditions were plasma power, 1500 W; carrier gas flow, 1 L/minute; makeup gas flow, 0.15 L/minute; sample depth, 8 mm; plasma gas, 15 L/minute. The concentrations were calculated against an external calibration curve with 50 \u0026micro;g/L of Ga used as the internal standard (IS) throughout (Gallium-71 isotope). Tissue samples (liver, spleen, lung, kidney, Intestine and pancreas) were suspended in 4 times the volume of analytical grade nitric acid, incubated at room temperature for up to 2 hours, followed by overnight digestion at 65\u003csup\u003e\u0026deg;\u003c/sup\u003eC. The samples were cooled and diluted 20-fold into the autosampler to reach a 10 mg/mL final concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell toxicity and uptake test\u003c/h2\u003e \u003cp\u003eUC-MSCs were cultured in MesenCult\u0026trade; media supplemented with 2.5% v/v human platelet lysate and 1% v/v penicillin-streptomycin (P/S). The CellTiter-Glo\u0026trade; assay was used to assess the cytotoxicity of the particles. Briefly, UC-MSCs were seeded at 10,000 cells per well in a 96-well clear bottom plate in the culture medium detailed above. Upon reaching 90% confluence, the cells were washed with PBS, serum-starved in MesenCult\u0026trade; media without platelet lysate for 4 h, and subsequently replaced with fresh MesenCult\u0026trade; media containing different concentrations of lipid-coated Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (0.5\u0026ndash;200 \u0026micro;g/mL). After incubation for 12 h, the CellTiter-Glo\u0026trade; assay was performed as per the manufacturer\u0026rsquo;s instructions. Untreated cells were used as controls. Fluorescence was measured using an IVIS Lumina II (Caliper Life Sciences) reader. Data was represented as fold change relative to the control. For determining the cellular uptake of the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, the UC-MSCs were seeded at 1.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well in 12-well clear-bottom plates and cultured to 90% confluency in MesenCult\u0026trade; media containing 2.5% human platelet lysate and 1% P/S. Subsequently, media containing FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs at a concentration of 10 \u0026micro;g/mL was added to each well and incubated for 2 h to 12 h. Following incubation, the cells were washed, fixed with 4% v/v paraformaldehyde (PFA), and the nuclei were stained with DAPI. The uptake of fluorescent particles was assessed using Celigo image cytometer (model number 200-BFFL-5C; Nexcelom Bioscience LLC, CA, USA). To visualize the localization of particles within the cells, confocal microscopy was used. Briefly, cells were seeded on a pre-inserted coverslip in cell culture plate wells. Following the attachment of cells, the particles were added at a concentration of 10 \u0026micro;g/mL. After incubation for 12 h, the cells were fixed in 4% PFA, the nuclei were stained with DAPI, and imaged using a confocal Leica DMi8 Inverted Microscope (Leica Microsystems Inc., Illinois, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. CT Imaging of particles\u003c/h2\u003e \u003cp\u003eTo assess Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs at relevant CT imaging levels with Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were dispersed in DI water with different mass concentrations ranging from 11.4, 22.8 45.5, 91.2, to 182.3 \u0026micro;g/mL based on the bismuth concentration. ICP-MS analyses of lipid-coated Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs suspension were shown with metal components: S\u0026thinsp;=\u0026thinsp;283.48 \u0026micro;g/mL, Au\u0026thinsp;=\u0026thinsp;440.11 \u0026micro;g/mL and Bi\u0026thinsp;=\u0026thinsp;3,647.83 \u0026micro;g/mL concentration. Phantoms were scanned using a small animal scanner, Siemens Inveon PET/CT system (Siemens Medical Solutions, Knoxville, TN USA) with scanning parameters listed in the next section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biodistribution\u003c/h2\u003e \u003cp\u003e Rats (8\u0026ndash;12 weeks old, male, Wistar) were purchased from Charles River Laboratories (USA) and housed according to Stanford University\u0026rsquo;s Administrative Panel for Laboratory Animal Care (APLAC). All procedures were performed in accordance with the regulations approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University. A total of 3 mL of lipid-coated FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs with an ICP-MS quantitative S\u0026thinsp;=\u0026thinsp;283.48 \u0026micro;g/mL, Au\u0026thinsp;=\u0026thinsp;440.11 \u0026micro;g/mL, Bi\u0026thinsp;=\u0026thinsp;3647.83 \u0026micro;g/mL concentration was injected intravenously (IV). Following the injection, the rats were anesthetized with isoflurane (3\u0026ndash;4% for induction and 1\u0026ndash;3% for maintenance) and imaged with CT after Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs administration using the Siemens Inveon PET/CT system (Siemens Medical Solutions, Knoxville TN USA). CT settings were a 121 projection and SB70 mm installed pallet. X-ray source conditions were applied for 79 keV tube voltage, 493 \u0026micro;A of tube current, 50 \u0026micro;M of spot size, 0.5 mm of filter, 18.37 X 12.2 cm of FOV, 2.45 of scale, 482.26-150.17, Hot metal of scale bar. Inveon Acquisitions workplace viewer 4.0 software was used to acquire data. All animals were euthanized 120 hours post-injection of the Au@Bi2S3 NRs, using carbon dioxide as the method of euthanasia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Histological analysis\u003c/h2\u003e \u003cp\u003eHistological analysis of the explanted organs (liver, spleen, pancreas, lung, and kidneys) at 120h following intravenous injection of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was performed to assess the biocompatibility of our particles in small animals. The excised organs were fixed in 10% formalin, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H\u0026amp;E). Furthermore, immunohistochemistry using CD45 staining to assess any inflammatory response to Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was performed, as previously described [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The stained sections were analyzed using a NanoZoomer slide scanner 2.0-RS (Hamamatsu Photonics, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs synthesis and characterization\u003c/h2\u003e \u003cp\u003eSolvothermal synthesis was used to synthesize Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs by mixing bismuth and sulfur precursors, such as thioacetamide and bismuth neodecanoate, dissolved in an organic solvent. The resulting solution was then placed in a high-pressure reactor autoclave and heated to the desired reaction temperature. As the bismuth and sulfur precursors react, they form bismuth sulfide, which precipitates as Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. The resulting mixture was then cooled, and Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were collected by centrifugation. Next, gold nanoparticles (AuNPs) were formed in situ on the surface of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs by reducing HAuCl\u003csub\u003e4\u003c/sub\u003e 3H\u003csub\u003e2\u003c/sub\u003eO with NaBH\u003csub\u003e4\u003c/sub\u003e to create Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. Here, gold ions, such as Gold (III) chloride trihydrate, were reduced onto the surface of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs using sodium borohydride. Biocompatible hydrophilic NPs were then developed with the help of a uniform lipid coating (DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e and phosphatidylcholine) using a film hydration method \u003cb\u003e(Schematic 1)\u003c/b\u003e; this reaction is carried out in an aqueous solution with the final Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs purified for characterization using high-resolution transmission electron microscopy (HR-TEM), scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), X-ray fluorescence (XRF), and inductively coupled plasma mass spectroscopy (ICP-MS).\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1\" name=\"Picture 1\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSchematic for the synthesis of Au@Bi\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eS\u003c/b\u003e \u003csub\u003e \u003cb\u003e3\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNRs.\u003c/b\u003e Multimodal Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs for high contrast X-ray CT imaging were obtained by core-shell structural synthesis. \u003cb\u003e(a)\u003c/b\u003e Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NR cores were synthesized using a solvothermal synthetic route. \u003cb\u003e(b)\u003c/b\u003e Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were stabilized with a lipid coating, consisting of phosphatidylcholine and DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e before then being coated with AuNPs on its surface to create Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 HR-TEM, SAED and STEM\u003c/h2\u003e \u003cp\u003eHR-TEM was used to examine the crystal structure and lattice defects of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, as well as their size, shape, and distribution. Low-power TEM images demonstrated that Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were of a uniform population having a rod shape of ~\u0026thinsp;10 nm width and ~\u0026thinsp;33\u0026ndash;43 nm length (N\u0026thinsp;=\u0026thinsp;20) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. HR-TEM images showed AuNPs with a uniform spherical size (~\u0026thinsp;3\u0026ndash;5 nm) that were homogeneously distributed and attached to the surface of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e\u003cb\u003e)\u003c/b\u003e. Furthermore, HR-TEM data also showed a lattice pattern with atoms at interplanar distances of 0.414 nm, 0.480 nm, 0.333 nm, and 0.260 nm, corresponding to the (220), (210), (130), and (311) planes, respectively (JCPDS 01-089-8963), which are in accordance with the atomic configuration of the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. The SAED patterns of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs are shown by simulation indexation. The simulation index lines correspond to the rod's interplanar spacing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. SAED confirmed that the AuNPs were coordinated with the bismuth template on the surface of the Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, which was assigned to the (111), (200), (220), (311), and (222) plane of gold (JCPDS; 04-007-4652; 03-065-3093 and 01-071-4073) for a face-centered cubic gold structure. The simulation indexation of the orthorhombic Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e SAED pattern shows planes of (020), (211), (040), (131), (311), (231), (331), (060), (610), and (630) (JCPDS; 01-089-8963; 04-019-2866) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], all of which confirm that Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were efficiently synthesized. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) illustrated the heterostructure and distribution of elements of bismuth (red), sulfur (blue), and gold (yellow) particles with mapping showing that the core of the particles was composed of Bi and S, while the shell was composed of Au (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Structural characterization: EDX and XRD\u003c/h2\u003e \u003cp\u003eEnergy dispersive X-ray spectroscopy (EDX) can identify the elemental composition of NPs by measuring the energy and intensity of X-rays emitted from the sample when it is bombarded with high-energy electrons; this provides detailed information about the structure and elemental composition of nanomaterials. EDX spectroscopy analysis demonstrated Bi, S, and Au signals with signature energy peaks detected in Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs \u003cb\u003e(Fig.\u0026nbsp;2a).\u003c/b\u003e It is important to note that EDX spectroscopy is a surface analysis technique that only provides information about the top few atomic layers of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. Hence, we also used XRD to obtain more detailed information about the crystal structure of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. Specifically, the XRD diffraction pattern was used to identify the presence of specific phases in the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and to determine the crystal structure of those phases. The crystal structure and lattice planes of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were assessed using powder XRD studies. The XRD pattern of plain (undecorated) Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs showed signature peaks at 23.16\u0026deg; (220), 24.29\u0026deg; (130), 25.71\u0026deg; (310), 28.11\u0026deg; (021), 29.19\u0026deg; (211), 32.39\u0026deg; (221), 33.50\u0026deg; (301), 34.50\u0026deg; (311), 36.40\u0026deg; (240), 38.63\u0026deg; (231), 40.77\u0026deg; (141), 43.30\u0026deg; (421), 46.10\u0026deg; (002), 47.16\u0026deg; (431), 53.29\u0026deg; (222), 54.9\u0026deg; (351), 55.3\u0026deg; (061), 55.42\u0026deg; (360), and 59.23\u0026deg; (242), corresponding to a d-spacing of 0.396 nm, 0.356 nm, 0.352 nm, 0.324 nm, 0.310 nm, 0.280 nm, 0.271 nm, 0.263 nm, 0.251 nm, 0.245 nm, 0.225 nm, 0.211 nm, 0.198 nm, 0.194 nm, 0.177 nm, 0.173 nm, 0.167 nm and 0.155 nm, respectively, all of which belong to the orthorhombic structure (JCPDS: 01-089-8963) of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. For Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, along with the Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs peaks as mentioned above, additional peaks at 25.26\u0026deg; (110), 36.40\u0026deg; (111) and 43.30\u0026deg; (200), were observed for the planes that correspond to 0.355 nm, 0.235 nm, and 0.203 nm, respectively (JCPDS: 04-007-4652; 01-071-4073). XRD patterns with no anonymous peaks indicated that the synthesized nanomaterials were free from contaminants \u003cb\u003e(Fig.\u0026nbsp;2b)\u003c/b\u003e. It is important to note that XRD is a non-destructive technique and does not provide information about the chemical composition of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. To determine the chemical composition of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, we used other techniques such as XPS, XRF spectrometer, and ICP-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Chemical characterization: XPS, XRF, and ICP-MS.\u003c/h2\u003e \u003cp\u003eXPS is a surface-sensitive analytical technique that measures the elemental composition and chemical states of the elements present at the surface of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (Fig.\u0026nbsp;2c-e). The survey spectrum revealed the presence of elements, such as Bi, S, O, and C, indicating the high purity of the particles. The peak for O arises, presumably, from adsorbed gases and/or oxides of C on the surfaces of the samples [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]; this observation is common in the case of ultrafine powder samples when exposed to atmospheric conditions. In XPS analysis of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, the main peaks observed are due to the Bi, S, and S2p orbitals and Bi 4f peaks. High-resolution XPS spectra of Au 4f and Bi 4f (Figs.\u0026nbsp;2d and 2e) further elucidated the composition of the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. For each asymmetric Bi 4f7/2 or Bi 4f5/2, the peak in both Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs could be deconvoluted into two peaks in the high-resolution XPS spectra in the Bi region (Fig.\u0026nbsp;2d). Peaks at 157.78 and 163.08 eV, which can be assigned to the binding energies of Bi 4f5/2 and Bi 4f7/2 in Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, respectively, belong to lattice Bi bound to S (Bi-S bond). However, in Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, both peaks had shifted toward a lower intensity, meaning some Au atoms in Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs may bind to S atoms to form an Au-S bond, which is in line with thiols demonstrating a high affinity for Au. The high-resolution XPS spectra in the Au region (Fig.\u0026nbsp;2e) showed two peaks at 85.88 and 89.68 eV, which can be assigned to the binding energies of Au 4f5/2 and Au 4f7/2 in Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, respectively, thereby indicating the presence of Au in the Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs nanostructures. The broad peak at around 224.68 eV corresponds to the binding energy of S2s (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e). \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e shows the peaks of S 2p3/2 and S 2p1/2 located at 165.58 and 160.28 eV, respectively, which match the literature values of the sulfide anion (S\u003csup\u003e2\u0026minus;\u003c/sup\u003e) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e). The observed values were found to be in close agreement with data reported by Grigas et al [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Collectively, the data showed that Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs have a uniform composition with bimetallic Bi/S cores and Au shells. Overall, XPS is a powerful tool for studying the surface chemistry of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and provides valuable information about the elemental composition of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e. In addition, the mass distribution of Au, Bi, and S was determined using XRF analysis. The Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs exhibited Bi/S\u0026thinsp;=\u0026thinsp;83.49/15.84 mass percent (%) and Bi/S/Au\u0026thinsp;=\u0026thinsp;74.53/16.69/8.77 mass percent (%), respectively (\u003cb\u003eFigure S2\u003c/b\u003e). Moreover, Bi/Au ratio\u0026thinsp;=\u0026thinsp;8.28:1% w/w content in lipid-decorated Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was determined by ICP-MS. These results indicate that the ratio of Bi/Au by ICP-MS and XRF matched.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Particle size and zeta potential analysis\u003c/h2\u003e \u003cp\u003eDynamic light scattering (DLS) was used to evaluate the average size, polydispersity, and zeta potential analysis of particles \u003cb\u003e(Fig.\u0026nbsp;2f)\u003c/b\u003e. The particles size of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were 224.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 nm and 206.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3nm, respectively. The particle size of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was ~\u0026thinsp;20 nm bigger than the size of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs because of the AuNPs decoration. The polydispersity index, which measures the sample's heterogeneity based on size, was comparable between the particles (0.2 and 0.3 for Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, respectively). Moreover, the DLS showed an average zeta potential of -26.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 mV for the Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and \u0026minus;\u0026thinsp;45.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 mV for Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, which demonstrates an increase of ~-20 mV for Au@B\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs compared to B\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. The higher negative surface charge of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was due to gold particles, which confer particle stability preventing any aggregation in aqueous solution. It is important to note that particle size and zeta potential are two important characteristics that can significantly affect biodistribution and stability [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"3\" name=\"Picture 3\"\u003e\u003c/div\u003e \u003cb\u003eFigure\u0026nbsp;2. Structural and surface chemistry characterization of particles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e Energy-dispersive X-ray (EDX) elemental mapping of Bi, S, and Au, with Cu originating from the TEM grid. \u003cb\u003e(b)\u003c/b\u003e X-ray diffraction (XRD) patterns of Bi₂S₃ NRs and Au@Bi₂S₃ NRs with indexed peaks corresponding to expected crystallographic planes. \u003cb\u003e(c\u0026ndash;e)\u003c/b\u003e X-ray photoelectron spectroscopy (XPS) analysis: \u003cb\u003e(c)\u003c/b\u003e full survey spectrum, \u003cb\u003e(d)\u003c/b\u003e Bi region, and \u003cb\u003e(e)\u003c/b\u003e Au region of Au@Bi₂S₃ NRs. \u003cb\u003e(f)\u003c/b\u003e Hydrodynamic size distribution of Bi₂S₃ NRs and Au@Bi₂S₃ NRs measured by dynamic light scattering (DLS).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Cellular uptake and toxicity evaluation\u003c/h2\u003e \u003cp\u003eTo evaluate the biocompatibility of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, we tested their cell uptake and toxicity using umbilical cord derived mesenchymal stem cells (UC-MSCs), given the potential to use this nanoparticle to label and track these cells. Fluorescence-based CellTiter-Glo\u0026trade; assays were used to test UC-MSC viability after exposure for 12 h to different concentrations of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (0.5 to 200 \u0026micro;g/mL). Viable cells showed fluorescence, while dead cells have a loss of fluorescence. It was noted that cell viability remained about 100% between 0.5\u0026ndash;40 \u0026micro;g/mL, but this drastically decreased at higher concentrations (60\u0026ndash;200 \u0026micro;g/mL) \u003cb\u003e(Figure S3)\u003c/b\u003e. Next, the effect of cell uptake of FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was studied. Representative confocal images of FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs (10 \u0026micro;g/mL) incubated with UC-MSCs for 12 h are reported in \u003cb\u003eFigure S3c\u003c/b\u003e, which shows green fluorescence in the cell cytoplasm (FITC staining of the particles) and blue nucleus (DAPI staining of cell nuclei). To further verify in vitro uptake of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs in UC-MSCs, FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs cellular uptake and intracellular localization of particles within UC-MSCs were assessed using a Celigo Imaging Cytometer in a time-series dynamic study (up to 12 h). Cellular uptake of FITC-Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was observed to occur in a time-dependent manner. Images show an increased amount of particle aggregates inside the cytoplasm of UC-MSCs over time (after 2h through 12h), as noted by the increased number of black particles or yellow dots in the bright-field and fluorescence images, respectively \u003cb\u003e(Figure S3d)\u003c/b\u003e. Particle uptake was relatively slow during the first few hours of incubation before reaching saturation at 10\u0026ndash;12 hours \u003cb\u003e(Figure S3d).\u003c/b\u003e It is important to note that the uptake of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs by UC-MSCs, as well as their potential effects on UC-MSCs, can vary depending on the specific study and the conditions used. Previous studies show that various factors such as the size, surface properties, concentration, and exposure time affect the particle uptake in cells and tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and their cellular uptake can occur through several mechanisms, including endocytosis, phagocytosis, and pinocytosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. In vivo biodistribution and clearance\u003c/h2\u003e \u003cp\u003eThe feasibility of utilizing Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs as a contrast agent for visualizing soft tissues was assessed through CT imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Initially, we compared the contrast efficacy of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs with that of mesoporous silica nanoparticles (SiNPs) in dry powder form (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Our results demonstrated that the combination of bismuth and gold in Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs produced significantly higher contrast levels compared to both Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs and SiNPs. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c).\u003c/p\u003e \u003cp\u003eNext, we investigated the in vivo biodistribution of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs following intravenous injection in rats, utilizing whole-body CT imaging with static scans conducted at various time points: 24, 48, 72, 96, and 120 hours post-injection. The data presented here focuses exclusively on the 24-hour time point, excluding information from the other time intervals. The 24-hour post-injection scan (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) revealed that Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs predominantly localized in major organs, including the liver, spleen, lung, and pancreas. This finding was further validated through ex vivo imaging of the harvested organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), which were collected at 120 hours after particle injection. Notably, the signal intensity at 120 hours in the liver and spleen was significantly stronger compared to that in the lung and pancreas, a trend corroborated by ICP-MS measurements. Additionally, ICP-MS results indicated that the bismuth content in various organs aligned with the signal intensity observed in CT imaging at the 120-hour mark. Accumulation of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs was confirmed in organs, with bismuth concentrations measured at 893.1\u0026thinsp;\u0026plusmn;\u0026thinsp;107.7 \u0026micro;g/g in the spleen, 714.4\u0026thinsp;\u0026plusmn;\u0026thinsp;118.1 \u0026micro;g/g in the liver, 126.4\u0026thinsp;\u0026plusmn;\u0026thinsp;22.5 \u0026micro;g/g in the lung, 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u0026micro;g/g in the pancreas, 74.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 \u0026micro;g/g in the kidney, and 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 \u0026micro;g/g in the intestine (\u003cb\u003eFigure S4\u003c/b\u003e). Hence, the extended imaging window following intravenous administration of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs presents significant potential for various clinical applications.\u003c/p\u003e \u003cp\u003eThe biodistribution and subsequent biological effects of NPs depend on a variety of factors, including their size, shape, core materials, and method of administration, to name a few. Following IV administration, Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, are predominantly taken up by the liver and spleen, as also shown by several other studies that have examined Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; this is in keeping with the function of organs of the reticuloendothelial system, of which the liver and spleen are part of, which remove and eliminate foreign substances from the bloodstream. Once in these organs, NPs are then processed. Here, there will be a balance between NPs clearance and potential ongoing inflammation (due to how the body processes the particles) that could result in potential local cellular injury [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Histological assessments\u003c/h2\u003e \u003cp\u003eThe liver, spleen, lung, kidney, and pancreas demonstrated normal gross size and morphology at the end of our study. Subjectively, Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs increased the darkness of the color of the liver and spleen compared to control animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e with H\u0026amp;E-stained samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) showing the particles evenly distributed throughout the parenchyma of each organ with no surrounding inflammatory cell infiltrate at 5 days. Moreover, fewer Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were seen in the lungs, pancreas, and kidney samples compared to the liver and spleen. Immunohistochemical staining, using CD45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), also indicated no leukocyte infiltration. Collectively, Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs elicited minimal-to-no inflammatory response in the short term. Future studies will aim to evaluate how these organs will handle Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs to determine their long-term safety profile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this work, we presented a simple technique to synthesize Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs via a solvothermal method and state-of-the-art characterization of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. By using a lipid-based phase changing agent (DSPE-PEG 2000) as a template for the functionalization of AuNPs onto Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, instead of Tween 20 that has been previously reported [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we have been able to control and optimize particle size, crystallinity, and reproducibility of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs. Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs were also seen to enhance soft tissue contrast on CT imaging of living animals and, hence, are promising nanoparticles for further development for both multimodal imaging as well as theranostic applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Histo-tech Laboratory Inc. for histology slide preparation and H\u0026amp;E staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.K. conceptualized, prepared, and characterized the nanorods; performed the in vivo experiments; conducted data collection and analysis and led manuscript preparation and writing. R.P. assisted with nanorod characterization, in vivo experiments, histology, data analysis, and manuscript preparation and editing. G.S. performed the cell uptake and toxicity experiments and data analysis. J.Z. edited and proofread the manuscript. A.S. acquired high-resolution transmission electron microscopy images and assisted with image analysis. R. Prasad draft overview and editing. B.D. edited and proofread the manuscript. J.W. performed the animal experiments. K.H. edited and proofread the manuscript. P.D. provided expert revisions. A.S.T. conceptualized and designed the experiments, supervised the project, and contributed to manuscript preparation and writing. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funding from the Department of Radiology at Stanford University, research grants from the NIDDK (R01DK129598, R01DK129343, R01DK141802), and the Akiko Yamazaki and Jerry Yang Faculty Scholarship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within this article and its Supplementary material. Raw data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all experiments in the present study were approved by the Administrative Panel on Laboratory Animal Care at Stanford University and were carried out per the Institutional Animal Care and Use Committee (IACUC) approval (Title: In vivo studies of engineered three-dimensional porous bio-scaffolds for pancreaticislet transplantation; Approval number: 32821; Last Approval date: 10/15/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAugustine R, Hasan A, Primavera R, Wilson RJ, Thakor AS, Kevadiya BD (2020) Mater Today Commun 25:101692. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mtcomm.2020.101692\u003c/span\u003e\u003cspan address=\"10.1016/j.mtcomm.2020.101692\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Kim J, Park YI, Lee N, Hyeon T (2018) ACS Cent Sci 4(3):324\u0026ndash;336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acscentsci.7b00574\u003c/span\u003e\u003cspan address=\"10.1021/acscentsci.7b00574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSukhanova A, Bozrova S, Sokolov P, Berestovoy M, Karaulov A, Nabiev I (2018) Nanoscale Res Lett 13(1):44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s11671-018-2457-x\u003c/span\u003e\u003cspan address=\"10.1186/s11671-018-2457-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKevadiya BD, Ottemann B, Mukadam IZ, Castellanos L, Sikora K, Hilaire JR, Machhi J, Herskovitz J, Soni D, Hasan M, Zhang W, Anandakumar S, Garrison J, McMillan J, Edagwa B, Mosley RL, Vachet RW, Gendelman HE (2020) Theranostics 10 (2), 630\u0026ndash;656. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/thno.39847\u003c/span\u003e\u003cspan address=\"10.7150/thno.39847\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel PR, De Jesus O (2022) CT Scan. In StatPearls, StatPearls Publishing Copyright \u0026copy; 2022, StatPearls Publishing LLC. Treasure Island (FL)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiwa S, Otsuka T, Orthop Sci J (2017) 22 (3), 391\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jos.2017.01.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jos.2017.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel M, Tann M, Liangpunsakul S (2021) Am J Med Sci 362(3):252\u0026ndash;259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.amjms.2020.10.031\u003c/span\u003e\u003cspan address=\"10.1016/j.amjms.2020.10.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePAUWELS E, VAN LOO D, CORNILLIE P, BRABANT L, VAN HOOREBEKE L (2013) J Microsc 250(1):21\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jmi.12013\u003c/span\u003e\u003cspan address=\"10.1111/jmi.12013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeh BM, FitzGerald PF, Edic PM, Lambert JW, Colborn RE, Marino ME, Evans PM, Roberts JC, Wang ZJ, Wong MJ, Bonitatibus PJ (2017) Jr Adv Drug Deliv Rev 113:201\u0026ndash;222. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.addr.2016.09.001\u003c/span\u003e\u003cspan address=\"10.1016/j.addr.2016.09.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabin O, Manuel Perez J, Grimm J, Wojtkiewicz G, Weissleder R (2006) Nat Mater 5(2):118\u0026ndash;122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nmat1571\u003c/span\u003e\u003cspan address=\"10.1038/nmat1571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndreucci M, Solomon R, Tasanarong A (2014) BioMed research international 2014, 741018\u0026ndash;741018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2014/741018\u003c/span\u003e\u003cspan address=\"10.1155/2014/741018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebb JA, Stacul F, Thomsen HS, Morcos SK (2003) Members of the *Contrast Media Safety Committee of the European Society of Urogenital Radiology. Eur Radiol 13(1):181\u0026ndash;184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00330-002-1650-5\u003c/span\u003e\u003cspan address=\"10.1007/s00330-002-1650-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh J, Daftary A (2008) J Nucl Med Technol 36(2):69\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2967/jnmt.107.047621\u003c/span\u003e\u003cspan address=\"10.2967/jnmt.107.047621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe F, Ji H, Feng L, Wang Z, Sun Q, Zhong C, Yang D, Gai S, Yang P, Lin J (2021) Biomaterials 264, 120453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biomaterials.2020.120453\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2020.120453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahan MM, Doiron AL (2018) Journal of Nanomaterials 2018, 5837276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2018/5837276\u003c/span\u003e\u003cspan address=\"10.1155/2018/5837276\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubramanyam P, Vinodkumar T, Deepa M, Subrahmanyam C (2019) J Mater Chem C 7(21):6398\u0026ndash;6405. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/C9TC00759H\u003c/span\u003e\u003cspan address=\"10.1039/C9TC00759H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerskovitz J, Hasan M, Machhi J, Mukadam I, Ottemann BM, Hilaire JR, Woldstad C, McMillan J, Liu Y, Seravalli J, Sarella A, Gendelman HE, Kevadiya BD (2021) Nanotheranostics 5 (4), 417\u0026ndash;430. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/ntno.59568\u003c/span\u003e\u003cspan address=\"10.7150/ntno.59568\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKevadiya BD, Woldstad C, Ottemann BM, Dash P, Sajja BR, Lamberty B, Morsey B, Kocher T, Dutta R, Bade AN, Liu Y, Callen SE, Fox HS, Byrareddy SN, McMillan JM, Bronich TK, Edagwa BJ, Boska MD, Gendelman HE (2018) Theranostics 8 (1), 256\u0026ndash;276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/thno.22764\u003c/span\u003e\u003cspan address=\"10.7150/thno.22764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElahi N, Kamali M, Baghersad MH (2018) Talanta 184, 537\u0026ndash;556. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.talanta.2018.02.088\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2018.02.088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabuzu D, Cirja A, Puiu R, Grumezescu AM (2015) Curr Top Med Chem 15(16):1605\u0026ndash;1613. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1568026615666150414144750\u003c/span\u003e\u003cspan address=\"10.2174/1568026615666150414144750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonavane G, Tomoda K, Makino K, Colloids, Surfaces B, Biointerfaces (2008) 66 (2), 274\u0026ndash;280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.colsurfb.2008.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2008.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss RD, Cole LE, Tilley JMR, Roeder RK (2014) Chem Mater 26(2):1187\u0026ndash;1194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/cm4035616\u003c/span\u003e\u003cspan address=\"10.1021/cm4035616\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L, Lu Y, Jiang C, Zhu Y, Yang X, Hu X, Lin Z, Zhang Y, Peng M, Xia H, Mao C (2018) Adv Funct Mater 28(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/adfm.201704623\u003c/span\u003e\u003cspan address=\"10.1002/adfm.201704623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAi K, Liu Y, Liu J, Yuan Q, He Y, Lu L (2011) Adv Mater 23(42):4886\u0026ndash;4891. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/adma.201103289\u003c/span\u003e\u003cspan address=\"10.1002/adma.201103289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Zheng X, Yan L, Zhou L, Tian G, Yin W, Wang L, Liu Y, Hu Z, Gu Z, Chen C, Zhao Y (2015) ACS Nano 9(1):696\u0026ndash;707. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/nn506137n\u003c/span\u003e\u003cspan address=\"10.1021/nn506137n\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X, Shi J, Bu Y, Tian G, Zhang X, Yin W, Gao B, Yang Z, Hu Z, Liu X, Yan L, Gu Z, Zhao Y (2015) Nanoscale 7(29):12581\u0026ndash;12591. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c5nr03068d\u003c/span\u003e\u003cspan address=\"10.1039/c5nr03068d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X, Shi J, Bu Y, Tian G, Zhang X, Yin W, Gao B, Yang Z, Hu Z, Liu X, Yan L, Gu Z, Zhao Y (2015) Nanoscale 7(29):12581\u0026ndash;12591. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c5nr03068d\u003c/span\u003e\u003cspan address=\"10.1039/c5nr03068d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Zhu J-J, Zhu J-M, Chen H-Y (2002) J Phys Chem B 106(15):3848\u0026ndash;3854. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jp0135003\u003c/span\u003e\u003cspan address=\"10.1021/jp0135003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrigas J, Talik E, Lazauskas V (2002) physica status solidi (b) 232 (2), 220\u0026ndash;230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/1521-3951(200208)232:2%3C220::AID-PSSB220%3E3.0.CO;2-F\u003c/span\u003e\u003cspan address=\"10.1002/1521-3951(200208)232:2%3C220::AID-PSSB220%3E3.0.CO;2-F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonavane G, Tomoda K, Makino K, Colloids Surf BB (2008) 66 (2), 274\u0026ndash;280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.colsurfb.2008.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2008.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDreaden EC, Austin LA, Mackey MA, El-Sayed MA (2012) Ther Deliv 3(4):457\u0026ndash;478. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4155/tde.12.21\u003c/span\u003e\u003cspan address=\"10.4155/tde.12.21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoshyar N, Gray S, Han H, Bao G (2016) 673\u0026thinsp;\u0026ndash;\u0026thinsp;92. Nanomed (Lond) 11(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2217/nnm.16.5\u003c/span\u003e\u003cspan address=\"10.2217/nnm.16.5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehzadi S, Serpooshan V, Tao W, Hamaly MA, Alkawareek MY, Dreaden EC, Brown D, Alkilany AM, Farokhzad OC, Mahmoudi M (2017) Chem Soc Rev 46(14):4218\u0026ndash;4244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c6cs00636a\u003c/span\u003e\u003cspan address=\"10.1039/c6cs00636a\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadrigilan S, Heydarpanahi F, Choupani J, Jaymand M, Samadian H, Hoseini-Ghahfarokhi M, Webster TJ, Tayebi L (2020) Int J Nanomed 15:7079\u0026ndash;7096. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/ijn.S250001\u003c/span\u003e\u003cspan address=\"10.2147/ijn.S250001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu M, Zheng J, Nano ACS (2015) 9 (7), 6655\u0026ndash;6674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acsnano.5b01320\u003c/span\u003e\u003cspan address=\"10.1021/acsnano.5b01320\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNel A, Xia T, M\u0026auml;dler L, Li N (2006) Science 311(5761):622\u0026ndash;627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1114397\u003c/span\u003e\u003cspan address=\"10.1126/science.1114397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDobrovolskaia MA, McNeil SE (2007) Nat Nanotechnol 2(8):469\u0026ndash;478. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nnano.2007.223\u003c/span\u003e\u003cspan address=\"10.1038/nnano.2007.223\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGustafson HH, Holt-Casper D, Grainger DW, Ghandehari H (2015) Nano Today 10(4):487\u0026ndash;510. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nantod.2015.06.006\u003c/span\u003e\u003cspan address=\"10.1016/j.nantod.2015.06.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Y, Chang Y, Feng Y, Jian H, Tang Z, Zhang H (2018) Angew Chem Int Ed 57(1):246\u0026ndash;251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/anie.201710399\u003c/span\u003e\u003cspan address=\"10.1002/anie.201710399\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrimavera R, Razavi M, Kevadiya BD, Wang J, Vykunta A, Di Mascolo D, Decuzzi P, Thakor AS (2021) Biofabrication 13 (3), 035011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1088/1758-5090/abdcac\u003c/span\u003e\u003cspan address=\"10.1088/1758-5090/abdcac\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CT imaging, Gold nanoparticles, Bismuth sulfide nanorods","lastPublishedDoi":"10.21203/rs.3.rs-8854864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8854864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of hybrid metal nanoparticles (NPs) for use as computed tomography (CT) contrast agents is a promising area of research. Achieving optimal in vivo performance of imaging for NPs is challenging and depends on their geometry, materials properties, bioreactivity, and biocompatibility. In this study, we designed and developed a novel CT contrast agent composed of gold nanoparticles (AuNPs) coordinated on the surface of bismuth sulfide-core nanorods (Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs) utilizing a solvothermal synthesis approach. We conducted solid-state characterization of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, demonstrating their structural configuration, excellent stability, uniformity, and high crystallinity. We also tested their biocompatibility with mesenchymal stem cells and found they exhibited no toxic effects. To evaluate the imaging potential of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs, we tested them in small animals using CT imaging. Our results showed contrast enhancement in soft tissues, indicating the retention of the particles at these locations with no local inflammatory responses. Taken together, our study provides a proof-of-concept for the robust synthesis and use of Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs as effective CT imaging contrast agents. Future work will explore the potential to functionalize Au@Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NRs with therapeutic molecules for theranostic applications.\u003c/p\u003e","manuscriptTitle":"Development of Gold-Bismuth Sulfide Nanorods (Au@Bi2S3 NRs) for X-ray- based imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 07:38:20","doi":"10.21203/rs.3.rs-8854864/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-09T07:46:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T04:23:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271225854227743009007567918365837787959","date":"2026-03-17T08:30:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T22:17:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249490584733540455204011350093294051913","date":"2026-03-09T14:19:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-08T21:30:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-02T04:21:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T05:16:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T17:27:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2026-02-25T17:21:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85667153-9acb-4126-8acc-0d7bd90fbb18","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T13:23:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 07:38:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8854864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8854864","identity":"rs-8854864","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}