Cheng-Liang Wang

Enhancement of cell radiation sensitivity by pegylated gold nanoparticles

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.

Optimizing the size and surface properties of polyethylene glycol (PEG)-gold nanoparticles by intense x-ray irradiation

The polyethylene glycol (PEG) modified gold nanoparticle complex was synthesized by a one-solution synchrotron x-ray irradiation method. The impact on the structure and morphology of the gold nanoparticles of process parameters such as the PEG molecular weight, the PEG/gold molar ratio and the x-ray dosage were investigated. The size of PEG modified gold particles was found to decrease with increasing PEG addition and x-ray dosage. With the capability to monitor the absorption spectra in situ during the fast synthesis process, this opens the way to accurate control of the size and distribution. PEG chains with an intermediate length (MW6000) were found optimal for size control and colloidal stability in biologically relevant media. Our x-ray synthesized PEG-gold nanoparticles could find interesting applications in nanoparticle-enhanced x-ray tumour imaging and therapy.

Full-field microimaging with 8 keV X-rays achieves a spatial resolutions better than 20 nm

Fresnel zone plates (450 nm thick Au, 25 nm outermost zone width) used as objective lenses in a full field transmission reached a spatial resolution better than 20 nm and 1.5% efficiency with 8 keV photons. Zernike phase contrast was also realized without compromising the resolution. These are very significant achievements in the rapid progress of high-aspect-ratio zone plate fabrication by combined electron beam lithography and electrodeposition.

Quantitative analysis of nanoparticle internalization in mammalian cells by high resolution X-ray microscopy

BackgroundQuantitative 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.ResultsPractical 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.ConclusionsWe 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.

Hard x-ray Zernike microscopy reaches 30 nm resolution

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

Enhancement of irradiation effects on cancer cells by cross-linked dextran-coated iron oxide (CLIO) nanoparticles

We investigated iron oxide nanoparticles with two different surface modifications, dextran coating and cross-linked dextran coating, showing that their different internalization affects their capability to enhance radiation damage to cancer cells. The internalization was monitored with an ultrahigh resolution transmission x-ray microscope (TXM), indicating that the differences in the particle surface charge play an essential role and dominate the particle-cell interaction. We found that dextran-coated iron oxide nanoparticles cannot be internalized by HeLa and EMT-6 cells without being functionalized with amino groups (the cross-linked dextran coating) that modify the surface potential from -18 mV to 13.4 mV. The amount of cross-linked dextran-coated iron oxide nanoparticles uptaken by cancer cells reached its maximum, 1.33 x 10(9) per HeLa cell, when the co-culture concentration was 40 microg Fe mL(-1) or more. Standard tests indicated that these internalized nanoparticles increased the damaging effects of x-ray irradiation, whereas they are by themselves biocompatible. These results could lead to interesting therapy applications; furthermore, iron oxide also produces high contrast for magnetic resonance imaging (MRI) in the diagnosis and therapy stages.

One-pot synthesis of AuPt alloyed nanoparticles by intense x-ray irradiation

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.

Imaging the cellular uptake of tiopronin-modified gold nanoparticles

AbstractWell-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. FigureA high resolution Transmission X-ray microscope image (A) captured the internalization and aggregation of tiopronin-coated Au nanoparticles in the vicinity of cell nucleus, the light dark area, of an EMG-6 cell. (B) One of the corresponding pictures produced by three-dimensional tomography reconstruction. The complete movie sequence of such pictures provides three-dimensional visual confirmation of the internalization and location of tiopronin-coated Au nanoparticles.

Tailored Au nanorods: optimizing functionality, controlling the aspect ratio and increasing biocompatibility

Monodisperse gold nanorods with high aspect ratio were synthesized by x-ray irradiation. Irradiation was first used to stimulate the creation of seeds. Afterward, nanorod growth was stimulated either by chemical reduction or again by x-ray irradiation. In the last case, the entire process took place without reducing agents. The shape of the final products could be controlled by modulating the intensity of the x-ray irradiation during the seed synthesis. In turn, the nanorod aspect ratio determines the absorption wavelength of the nanorods that can thus be optimized for different applications. Likewise, the aspect ratio influences the uptake of the nanorods by HeLa cells.

Controlled hydrogel photopolymerization inside live systems by X-ray irradiation

We successfully demonstrate the in situ formation by X-ray irradiation induced polymerization of a three-dimensional (3D) hydrogel based on poly(ethylene glycol) (PEG) diacrylate (PEG DA), with the option of heparin incorporation to enhance cell adhesion. The polymerization reaction can be completed in tens of seconds, thanks to the fast and uniform X-ray induced process. Eliminating polymerization initiators and the residual unreacted monomers improves the biocompatibility and stability of the hydrogel. The photopolymerization mechanism can be locally controlled by using a trimmed or a focused X-ray beam to allow precise in vivo administration deep in the tissue, without surgery. We also demonstrate that photopolymerized hydrogels containing polyethylenimine (PEI)/nucleic acid (DNA or small interfering RNA, siRNA) nanoparticles (NPs) can deliver such nanoparticles. The hydrogel is biocompatible with mice and is not toxic to epithelial cells.