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

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.

Free-electron-laser coherent diffraction images of individual drug-carrying liposome particles in solution.

Using the excellent performances of a SACLA (RIKEN/HARIMA, Japan) X-ray free electron laser (X-FEL), coherent diffraction imaging (CDI) was used to detect individual liposome particles in water, with or without inserted doxorubicin nanorods. This was possible because of the electron density differences between the carrier, the liposome, and the drug. The result is important since liposome nanocarriers at present dominate drug delivery systems. In spite of the low cross-section of the original ingredients, the diffracted intensity of drug-free liposomes was sufficient for spatial reconstruction yielding quantitative structural information. For particles containing doxorubicin, the structural parameters of the nanorods could be extracted from CDI. Furthermore, the measurement of the electron density of the solution enclosed in each liposome provides direct evidence of the incorporation of ammonium sulphate into the nanorods. Overall, ours is an important test for extending the X-FEL analysis of individual nanoparticles to low cross-sectional systems in solution, and also for its potential use to optimize the manufacturing of drug nanocarriers.