30 research outputs found
Quantitative analysis of nanoparticle internalization in mammalian cells by high resolution X-ray microscopy
<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p
Low temperature thermal dependent Filgrastim adsorption behavior detected with ToF-SIMS
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) detected changes in Filgrastim (granulocyte colony stimulating growth factor, G-CSF) adsorption behavior at a solid interface when exposed to temperatures as low as 35 °C, i.e., before thermal denaturation, was detected by circular dichroism (CD) or dynamic light scattering (DLS). Biopharmaceuticals rely on maintaining sufficient conformation to impart correct biological function in vivo. Stability of such molecules is critical during synthesis, storage, transport, and administration. CD analysis indicated loss of structure at temperatures greater than 60 °C, while DLS detected aggregation at 42 °C. Furthermore, we demonstrate the nature of G-CSF interaction with a surface was altered rapidly and at relatively low temperatures. Specifically, after 10 min thermal treatment, changes in adsorption behavior occurred at 35 °C indicated by principal component analysis of spectra as primarily due to increasing yields of methionine fragments. This was likely to be due to either altering the preferential protein orientation upon adsorption or greater denaturation exposing the hydrophobic core. This investigation demonstrates the sensitivity of ToF-SIMS in studying biopharmaceutical adsorption and conformational change and can assist with studies into promoting their stability.
Imaging the penetration and distribution of zinc and zinc species after topical application of zinc pyrithione to human skin
Zinc pyrithione is an active component incorporated in an extensive range of topically applied commercial products that are used worldwide. Despite its prevalence, no published study has investigated the penetration of zinc from the zinc pyrithione complex into human skin. Zinc is crucial for healthy skin function however an elevated concentration of labile zinc is toxic outside a narrow concentration range. Synchrotron X-ray fluorescence microscopy in conjunction with X-ray absorption near edge structure spectroscopy was used to map the deposition of zinc, quantitate the amount of zinc within the skin and to identify a change in the chemical form of zinc after application. This study has demonstrated a similar to 3.8 fold increase in zinc concentration within the viable epidermis (VE) after 24 h topical application of zinc pyrithione that increased significantly by similar to 250 fold after 48 h when compared to control skin. Confocal microscopy using a labile zinc specific dye, ZinPyr-1, showed that zinc pyrithione disrupted the skin cells zinc homeostasis and significantly increased the intracellular zinc concentration leading to cell toxicity. Overall, this study demonstrates that topical application of zinc pyrithione formulations leads to an increase in zinc penetration in human skin, consequently, raising concerns for potential localised toxicity to occur
Synchrotron XPS studies of collector adsorption and co-adsorption on gold and gold: silver alloy surfaces
Penetration of Zinc into Human Skin after Topical Application of Nano Zinc Oxide Used in Commercial Sunscreen Formulations
Zinc oxide nanoparticles (ZnO NPs) are a key constituent of many commercial broad-spectrum sunscreens. Studies have shown that these NPs are retained on the superficial layers of the skins' barrier layer, the stratum corneum, and solubilized zinc species from the ZnO NPs have been shown qualitatively to penetrate intact human skin. The cytotoxicity of zinc is concentration- and species-dependent; however, to date, the amount of zinc permeating the skin strata is yet to be determined. Here, we applied commercial ZnO NPs to intact and impaired ex vivo barrier human skin. Artificial human sweat (to provide an electrolyte solution) and caprylic capric triglyceride (CCT; a common sunscreen formulation base) suspensions were applied to encompass potential "in-use"scenarios. A state-of-the-art multimodal approach analyzed zinc permeation. Our data show that elevated zinc concentrations within the skin are dependent on a number of variables, with barrier impairment and time being the most important factors followed by the vehicle, where sweat was more impactful than CCT. When ZnO NPs were applied to impaired barrier skin for 24 h, there was a 60-65-fold increase in zinc in the viable epidermis for both CCT and sweat compared to the control, increasing >100-fold after 48 h. Importantly, we identify that the localized cutaneous zinc concentration increase is not present as the nano ZnO that is used in sunscreens but only after dissolution and permeation as a different solubilized zinc species
Quantitative synchrotron X-ray fluorescence study of the penetration of transferrin-conjugated gold nanoparticles inside model tumour tissues
The next generation of therapeutic nanoparticles in the treatment of cancer incorporate specific targeting. There is implicit importance in understanding penetration of targeted nanomedicines within tumour tissues via accurate and quantitative temporospatial measurements. In this study we demonstrate the potential of state-of-the-art synchrotron X-ray fluorescence microscopy (XFM) to provide such insights. To this end, quantitative mapping of the distribution of transferrin-conjugated gold nanoparticles inside multicellular tumour spheroids was achieved using XFM and compared with qualitative data obtained using reflectance confocal microscopy. Gold nanoparticles conjugated with human transferrin with a narrow size-distribution and high binding affinity to tumour cells were prepared as confirmed by cellular uptake studies performed on 2D monolayers. Although the prepared 100 nm transferrin-conjugated gold nanoparticles had high targeting capability to cancer cells, penetration inside multicellular spheroids was limited even after 48 hours as shown by the quantitative XFM measurements. The rapid, quantitative and label-free nature of state-of-the-art synchrotron XFM make it an ideal technology to provide the structureâactivity relationship understanding urgently required for developing the next generation of immuno-targeted nanomedicines
Cu(In1-xGax)S-2 nanocrystals and films: low-temperature synthesis with size and composition control
We demonstrate a single-step X-ray irradiation process that yields high-quality Cu(In1-xGax)S-2 nanocrystals in colloidal solutions, with complete control of size and composition. Thin films produced by drop-casting exhibit high-quality photoresponse, confirming that our process is suitable for microelectronics applications
Fabrication of single crystal CuGaS2 nanorods by X-ray irradiation
CuGaS2 nanorods were synthesized by irradiating the precursor solution with intense X-rays. The products are single crystal nanorods with preferential [220] growth and a uniform size distribution. We also report on the photoresponse of drop-cast films of these nanorods
Unhindered copper uptake by glutaraldehyde-polyethyleneimine coatings in an artificial seawater model system with adsorbed swollen polysaccharides and competing ligand EDTA
<p>Shortly after a surface is submerged in the sea, a conditioning film is generally formed by adsorption of organic molecules, such as polysaccharides. This could affect transport of molecules and ions between the seawater and the surface. An artificial seawater model system was developed to understand how adsorbed polysaccharides impact copper binding by glutaraldehyde-crosslinked polyethyleneimine coatings. Coating performance was also determined when competed against copper-chelating EDTA. Polysaccharide adsorption and copper binding and distribution were investigated using advanced analytical techniques, including depth-resolved time-of-flight secondary ion mass spectroscopy, grazing incidence X-ray absorption near-edge spectroscopy, quartz crystal microbalance with dissipation monitoring and X-ray photoelectron spectroscopy. In artificial seawater, the polysaccharides adsorbed in a swollen state that copper readily penetrated and the glutaraldehyde-polyethyleneimine coatings outcompeted EDTA for copper binding. Furthermore, the depth distribution of copper species was determined with nanometre precision. The results are highly relevant for copper-binding and copper-releasing materials in seawater.</p
X ray fluorescence in member states
Synchrotron light sources have provided highly versatile X ray sources for XRF mapping. The intensity, polarization and monochromatic nature provide several benefits for studying trace level elemental distributions in low Z materials such as biological samples. The greater intensity and collimation enable efficient focusing for small probing beam size. The polarization reduces detected scatter and the monochromatic incident beam reduces spectral background which improves signal to noise and detection limits. The ability to tune the incident energy also allows for easy distinction of elements with overlapping emission lines by selectively determining which elements are excited while omitting others. This can also be utilized to further optimize detection limits or conduct X ray absorption spectroscopy