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An original synthesis method based on X-ray irradiation produced gold nanoparticles (AuNPs) with two important properties for biomedical research: intense visible photoluminescence and very high accumulation in cancer cells. The nanoparticles, coated with MUA (11-mercaptoundecanoid acid), are very small (1.4 nm diameter); the above two properties are not present for even slightly larger sizes. The small MUA-AuNPs are non-cytotoxic (except for very high concentrations) and do not interfere with cancer cell proliferation. Multimodality imaging using visible light fluorescence and X-ray microscopy is demonstrated by tracing the nanoparticle-loaded tumor cells. (C) 2012 Elsevier Inc. All rights reserved.

We demonstrate that, with appropriate staining, high-resolution X-ray microscopy can image complicated tissue structures—cerebellum and liver—and resolve large or small amounts of Au nanoparticles in these tissues. Specifically, images of tumor tissue reveal high concentrations of accumulated Au nanoparticles. PEG (poly(ethylene glycol)) coating is quite effective in enhancing this accumulation and significantly modifies the mechanism of uptake by reticuloendothelial system (RES) organs. Refereed/Peer-reviewed

We describe a simple and effective method to obtain colloidal surface-functionalized Au nanoparticles. The method is primarily based on irradiation of a gold solution with high-flux X-rays from a synchrotron source in the presence of 11-mercaptoundecanoic acid (MUA). Extensive tests of the products demonstrated high colloidal density as well as excellent stability, shelf life, and biocompatibility. Specific tests with X-ray diffraction, UV-visible spectrometry, visible microscopy, Fourier transform infrared spectroscopy, dark-field visible-light scattering microscopy, and transmission electron microscopy demonstrated that MUA, being an effective surfactant, not only allows tunable size control of the nanoparticles, but also facilitates functionalization. The nanoparticle sizes were 6.45 ± 1.58, 1.83 ± 1.21, 1.52 ± 0.37 and 1.18 ± 0.26 nm with no MUA and with MUA-to-Au ratios of 1:2, 1:1, and 3:1. The MUA additionally enabled functionalization with l-glycine. We thus demonstrated flexibility in controlling the nanoparticle size over a large range with narrow size distribution. Refereed/Peer-reviewed

Well-dispersed gold nanoparticles (NP) coated with tiopronin were synthesized by X-ray irradiation without reducing agents. High-resolution transmission electron microscopy shows that the average core diameters of the NPs can be systematically controlled by adjusting the tiopronin to Au mole ratio in the reaction. Three methods were used to study the NP uptake by cells: quantitative measurements by inductively coupled plasma mass spectrometry, direct imaging with high lateral resolution transmission electron microscopy and transmission X-ray microscopy. The results confirmed that the NP internalization mostly occurred via endocytosis and concerned the cytoplasm. The particles, in spite of their small sizes, were not found to arrive inside the cell nuclei. The synthesis without reducing agents and solvents increased the biocompatibility as required for potential applications in analysis and biomedicine in general Refereed/Peer-reviewed

The spatial distribution of X-ray synthesized, PEG coated Au nanoparticles in cancer-bearing mice and their time dependent accumulation were investigated with inductive coupled plasma – optical emission spectroscopy (ICP-OES), transmission electron microscopy (TEM) and histological imaging. The results conclusively demonstrate that the particles strongly accumulate in tumor regions; up to ∼25 times more than in normal muscle tissue. This accumulation increases with the time after injection for up to ∼12 h in tumor, spleen and liver tissues, whereas for most non-tumor regions it saturates or decreases (blood, lung, brain, heart, and kidney). The impact of this result is discussed with special emphasis on passive targeted drug delivery and could also be used for the delineation and early-stage imaging of small tumors. Refereed/Peer-reviewed

Au/TiO2 nanocomposite particles were synthesized by a method based on intense X-ray irradiation without adding any reducing agent or stabilizer. The nanocomposite exhibits promising photocatalytic and biological properties at physiologically relevant concentration ([Au] = 0.028 mM, [TiO2] = 0.5 mM). The structure and photocatalysis were examined by X-ray diffraction, electron microscopy and ultraviolet-visible spectroscopy demonstrating that gold nanoparticles of 2-5 nm size were successfully deposited on TiO2 nanoparticle surfaces. The nanocomposite exhibited good colloidal stability within a typical cellular environment and was nontoxic to cancer cell according to evaluations under controlled conditions. The Au/TiO2 nanoparticles were also found to enhance the photocatalytic efficiency of UV radiation and even more that of X-ray radiation. In vitro studies indicated that the cell-killing effect under X-ray irradiation is more pronounced with the addition of Au/TiO2 nanoparticles than of bare TiO2 nanoparticles. (C) 2009 Elsevier B.V. All rights reserved.

Reductant, stabilizer-free colloidal gold solutions were fabricated by a new room-temperature synchrotron X-ray irradiation method. The influence of process parameters such as the pH value and the exposure time on the structure of gold nanoparticles was investigated. The mechanisms underlying the X-ray-triggered reduction of gold ions and the formation of gold clusters are discussed in detail. The X-ray irradiation derived highly concentrated gold nanoparticles are readily to be re-dispersed and possess suitable colloidal stability within cellular environment. The characterization included a study of the possible cytotoxicity for the EMT-6 tumor cell line: the negative results indicate that the gold clusters produced with our approach are biocompatible.

The formation of colloidal unmodified (naked) gold nanoparticles is investigated by irradiation of a precursor solution with X-rays from a synchrotron source. An interesting morphological evolution as a function of exposure time, from cross-linked network-like structure to individual particles, has been discovered. The particle size decreased with the exposure time and was influenced by the ionic strength of the precursor solution. Contrary to gamma-ray exposure, an OH radical scavenger was not required for cluster formation.

Background Nanoparticles can be used for targeted drug delivery, in particular for brain cancer therapy. However, this requires a detailed analysis of nanoparticles from the associated microvasculature to the tumor, not easy because of the required high spatial resolution. The objective of this study is to demonstrate an experimental solution of this problem, based in vivo and post-mortem whole organ imaging plus nanoscale 3-dimensional (3D) X-ray microscopy. Results The use of gold nanoparticles (AuNPs) as contrast agents paved the way to a detailed high-resolution three dimensional (3D) X-ray and fluorescence imaging analysis of the relation between xenografted glioma cells and the tumor-induced angiogenic microvasculature. The images of the angiogenic microvessels revealed nanoparticle leakage. Complementary tests showed that after endocytotic internalization fluorescent AuNPs allow the visible-light detection of cells. Conclusions AuNP-loading of cells could be extended from the case presented here to other imaging techniques. In our study, they enabled us to (1) identify primary glioma cells at inoculation sites in mice brains; (2) follow the subsequent development of gliomas. (3) Detect the full details of the tumor-related microvasculature; (4) Finding leakage of AuNPs from the tumor-related vasculature, in contrast to no leakage from normal vasculature. Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0140-2) contains supplementary material, which is available to authorized users.

This study was designed to demonstrate the potential of small nanoparticulate lymphotropic contrast agents designed to bind with high affinity to lymphoid cells overexpressing the CD45 antigen. To this end, small gold nanoparticles used as model were conjugated to anti-CD45 antibodies and injected in mice in the dorsal toe of the fore/hind paw. Chemical analysis demonstrated rapid uptake and transport of the nanoparticles in the lymphatic as well as significant retention of the nanoparticles with high bind- ing affinity to lymphoid cells in the popliteal and axillary lymph nodes in comparison to non-targeted nanoparticles. Refereed/Peer-reviewed

Background Angiogenesis is widely investigated in conjunction with cancer development, in particular because of the possibility of early stage detection and of new therapeutic strategies. However, such studies are negatively affected by the limitations of imaging techniques in the detection of microscopic blood vessels (diameter 3-5 μm) grown under angiogenic stress. We report that synchrotron-based X-ray imaging techniques with very high spatial resolution can overcome this obstacle, provided that suitable contrast agents are used. Results We tested different contrast agents based on gold nanoparticles (AuNPs) for the detection of cancer-related angiogenesis by synchrotron microradiology, microtomography and high resolution X-ray microscopy. Among them only bare-AuNPs in conjunction with heparin injection provided sufficient contrast to allow in vivo detection of small capillary species (the smallest measured lumen diameters were 3-5 μm). The detected vessel density was 3-7 times higher than with other nanoparticles. We also found that bare-AuNPs with heparin allows detecting symptoms of local extravascular nanoparticle diffusion in tumor areas where capillary leakage appeared. Conclusions Although high-Z AuNPs are natural candidates as radiology contrast agents, their success is not guaranteed, in particular when targeting very small blood vessels in tumor-related angiography. We found that AuNPs injected with heparin produced the contrast level needed to reveal--for the first time by X-ray imaging--tumor microvessels with 3-5 μm diameter as well as extravascular diffusion due to basal membrane defenestration. These results open the interesting possibility of functional imaging of the tumor microvasculature, of its development and organization, as well as of the effects of anti-angiogenic drugs.

Gold nanoparticles (GNPs) are intravenously administered to mice. Deposition at the pilosebacious unit and whiskers is visualized with X-ray fl uorescence after 30 minutes and 14 days. After 30 minutes the dermal papilla, bulge region, and root sheath all contain NPs. GNPs are driven externally out from follicles, counteractive to transfollicular delivery. After 14 days, gold bands in hairs refl ect pharmacokinetic profi les indicating blood concentration kinetics. Elimination rate constants infer half-lives from 3 hairs from an individual mouse within reasonable agreement (6.08, 7.15, and 8.66 hours). 3D reconstruction of NP distributions with confocal microscopy identifi es aggregates within the medullary canal. Intermittent NP deposition continues randomly over the two week period demonstrating prolonged NP mobility in vivo. NPs are still retained at the hair bulb after 14 days. The observations further account for the excretory mechanisms of NPs and their behavior in the pilosebacous unit, and demonstrate monitoring pharmacokinetic behavior in individual animals. Refereed/Peer-reviewed

We show that sufficient concentrations of gold nanoparticles produced by an original synthesis method in EMT-6 and CT-26 cancer cells make it possible to detect the presence, necrosis and proliferation of such cells after inoculation in live mice. We first demonstrated that the nanoparticles do not interfere with the proliferation process. Then, we observed significant differences in the tumor evolution and the angiogenesis process after shallow and deep inoculation. A direct comparison with pathology optical images illustrates the effectiveness of this approach Refereed/Peer-reviewed

We explored a very interesting gold nanoparticle system?pegylated gold in colloidal solution?and analyzed its uptake by mice colorectal adenocarcinoma CT26 tumor cells and the impact on the cell's response to x-ray irradiation. We found that exposure to polyethylene glycol (PEG) modified ('pegylated') 4.7 ? 2.6?nm gold nanoparticles synthesized by a novel synchrotron-based method enhances the response of CT26 cells to x-ray irradiation. Transmission electron microscopy (TEM) and confocal microscopy revealed that substantial amounts of such nanoparticles are taken up and absorbed by the cells and this conclusion is supported by quantitative induced coupled plasma (ICP) results. Standard tests indicated that the internalized particles are highly biocompatible but strongly enhance the cell damage induced by x-ray irradiation. Synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy analyzed the chemical aspects of this phenomenon: the appearance of C = O stretching bond spectral features could be used as a marker for cell damage and confirmed the enhancement of the radiation-induced toxicity for cells.

Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

We present an effective solution for the problem of contrast enhancement in phase-contrast microangiography, with the specific objective of visualising small(

We report the analysis of a reducing-agent-free synthetic method based on intense X-ray irradiation in the presence of 11-mercaptoundecanoic acid (MUA) and their application in biomedical imaging. The method is very effective in controlling the size and size distribution of Au nanoparticles in colloidal solutions: gold nanoparticles (AuNPs) as small as ∼1.2 ± 0.25 nm are synthesized in a one-pot irradiation procedure without reducing agents. We found two coexisting properties of these small AuNPs: a strong visible fluorescence and very high accumulation in cells, which enabled a new type of multimodality imaging using X-ray and visible light microscopies. These properties come as a direct consequence of the precise size control; specifically, they exist only when the particle size is smaller than ∼1.4 nm. We found that this process critically depends on several parameters including the solution pH, the solution temperature and the irradiation time. Most important is the fact that the increase of the irradiation time is not necessarily beneficial, since an optimum value exists to obtain small particles with a narrow size distribution. The 1.4 nm AuNPs are not toxic and significantly accumulate in tumor tissues in animal tests, as confirmed by X-ray imaging and fluorescence microscopy. This biodistribution could open the way for the use of these nanoparticles in X-ray imaging. Refereed/Peer-reviewed

We synthesized AuPt alloyed nanoparticles in colloidal solution by a one-pot procedure based on synchrotron x-ray irradiation in the presence of PEG (polyethylene glycol). The exclusive presence of alloyed nanoparticles with fcc structure was confirmed by several different experiments including UV–vis spectroscopy, x-ray diffraction (XRD) and transmission electron microscopy (TEM). The composition of the AuPt alloyed nanoparticles can be varied in a continuous fashion by simply varying the feed ratios of Au and Pt precursors. The nanoparticles exhibited colloidal stability and biocompatibility, important for potential applications. Refereed/Peer-reviewed

Highly concentrated gold nanoparticles (20 ± 5 nm) were produced by an x‐ray irradiation method. The particles were then examined for the interactions between gold and tumor cells under x‐ray radiation conditions. The biological effects of gold nanoparticles were investigated in terms of the internalization, cytotoxicity and capability to enhance x‐ray radiotherapy. The results of this investigation indicated that x‐ray derived gold nanoparticles were nontoxic to CT‐26 cell line and immobilized within cytoplasm. The irradiation experiments provided further evidence that gold nanoparticles were capable of enhancing the efficiency of radiotherapy.

Background Quantitative analysis of nanoparticle uptake at the cellular level is critical to nanomedicine procedures. In particular, it is required for a realistic evaluation of their effects. Unfortunately, quantitative measurements of nanoparticle uptake still pose a formidable technical challenge. We present here a method to tackle this problem and analyze the number of metal nanoparticles present in different types of cells. The method relies on high-lateral-resolution (better than 30 nm) transmission x-ray microimages with both absorption contrast and phase contrast -- including two-dimensional (2D) projection images and three-dimensional (3D) tomographic reconstructions that directly show the nanoparticles. Results Practical tests were successfully conducted on bare and polyethylene glycol (PEG) coated gold nanoparticles obtained by x-ray irradiation. Using two different cell lines, EMT and HeLa, we obtained the number of nanoparticle clusters uptaken by each cell and the cluster size. Furthermore, the analysis revealed interesting differences between 2D and 3D cultured cells as well as between 2D and 3D data for the same 3D specimen. Conclusions We demonstrated the feasibility and effectiveness of our method, proving that it is accurate enough to measure the nanoparticle uptake differences between cells as well as the sizes of the formed nanoparticle clusters. The differences between 2D and 3D cultures and 2D and 3D images stress the importance of the 3D analysis which is made possible by our approach.