Environ. Sci. Technol. 2006, 40, 4353-4359
Interaction of Fine Particles and Nanoparticles with Red Blood Cells Visualized with Advanced Microscopic Techniques† B A R B A R A M . R O T H E N - R U T I S H A U S E R , * ,‡ SAMUEL SCHU ¨ RCH,§ BEAT HAENNI,‡ NADINE KAPP,| AND PETER GEHR‡ Institute of Anatomy, Division of Histology, University of Bern, Bern, Switzerland, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada, and Department of Veterinary Anatomy, University of Bern, Bern, Switzerland
So far, little is known about the interaction of nanoparticles with lung cells, the entering of nanoparticles, and their transport through the blood stream to other organs. The entering and localization of different nanoparticles consisting of differing materials and of different charges were studied in human red blood cells. As these cells do not have any phagocytic receptors on their surface, and no actinmyosin system, we chose them as a model for nonphagocytic cells to study how nanoparticles penetrate cell membranes. We combined different microscopic techniques to visualize fine and nanoparticles in red blood cells: (I) fluorescent particles were analyzed by laser scanning microscopy combined with digital image restoration, (II) gold particles were analyzed by conventional transmission electron microscopy and energy filtering transmission electron microscopy, and (III) titanium dioxide particles were analyzed by energy filtering transmission electron microscopy. By using these differing microscopic techniques we were able to visualize and detect particles e0.2 µm and nanoparticles in red blood cells. We found that the surface charge and the material of the particles did not influence their entering. These results suggest that particles may penetrate the red blood cell membrane by a still unknown mechanism different from phagocytosis and endocytosis.
Introduction There is evidence from a number of epidemiological studies that particulate matter causes adverse health effects associated with increased pulmonary and cardiovascular mortality (1, 2). Recent studies indicate a specific toxicological role of nanoparticles (3). Thus particles a few nanometers in diameter are of particular interest, but hardly any information is available today on their effects on cells, tissues, and organs. In addition to the generation from combustion processes of nanoparticles in large amounts, there are progressively more nanoparticles released into the air, water, and soil every year from other sources, especially those related to nanotech* Corresponding author e-mail:
[email protected]; phone: ++41-31-631-8441; fax: ++41-31-631-3807. † This paper is part of a focus group on Effects of Nanoparticles. ‡ Institute of Anatomy, Division of Histology, University of Bern. § University of Calgary. | Department of Veterinary Anatomy, University of Bern. 10.1021/es0522635 CCC: $33.50 Published on Web 04/05/2006
2006 American Chemical Society
nological processes (4, 5). Nanoparticles have unique chemical, physical, and electrical properties. They behave unlike solids, liquids, or gases, but they share characteristics of fine particles and solutes. New properties emerge that are not exhibited by larger particles having the same chemical composition. These properties include different colors and different electronic, magnetic, and mechanical properties, any or all of which may be altered at the nanoscale. It is very important to collect risk data, in particular health risk data, so that questions can be answered and problems can be addressed during the early stage of the development of the new technologies (6). Most of the concerns regarding nanomaterials are due to the fact that nanoparticles are more inflammatory and toxic than fine particles (7). Not only is the effect of nanoparticles on human health an important issue, but also in particular the mechanisms involved in the penetration of the human body by these particles. The wall of the respiratory system consists of a series of barrier components that protect against foreign material. These include the surfactant film (8-10), the mucociliary system (11), highly phagocytic airway macrophages (12-14), and the epithelium with its tight junctions (15). However, despite the existence of these barriers, respiratory diseases are frequent and increasing (16) and more attention is being directed toward elucidating how and when the antigens evade these barriers. All inhalable particles (particulate matter 0.2 µm) are always observed outside the cells (d; black arrow). range, it is difficult to improve the signal-to-noise ratio due to very low intensities. Since the elemental analysis of silver is easier, gold particles were enhanced with silver. Silver (and therefore gold particles) could easily be detected within cells (Figure 3c). Silver was also detected in larger complexes attached to the cells or as free aggregates (Figure 3d).
Discussion The visualization of small objects in cells is very challenging. On one hand the resolution of light microscopy is about 0.2 µm at best depending on the wavelength of the light. On the other hand, by using TEM, although the resolution is much higher, the particles are very difficult to identify, as the size of the objects is similar to that of, for example, ribosomes. By using a variety of advanced microscopic techniques we were able to visualize nanoparticles with different surface charges and of different material with light as well as with transmission electron microscopy. By combining these methods the identification and localization of nanoparticles is significantly better than by using either method alone. Using light microscopy we can study many cells in a short time, however, the detailed intracellular localization (micro4356
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or nanoenvironment) has to be examined with electron microscopy, a time-consuming method where only few cells can be studied. Fine particles (1 and 0.2 µm) and nanoparticles (0.078 and 0.02 µm) which were fluorescently labeled and made of a polystyrol material have been studied in RBC by LSM combined with digital image restoration. To overcome limitations caused by out-of-focus components resulting in blur and noise, approaches have been developed to mathematically reverse the degrading effects by deconvolution algorithms (30, 31). Most deconvolution algorithms require the knowledge of the point spread function, which is defined for linear systems as the image of a point source of the microscope (for a review see 32). For every study the difference between the numerically determined (theoretical) point spread function and the measured one has to be compared (28). We have compared the two approaches in this study and did not find a discernible difference (data not shown). We then decided to use the theoretical point spread function as a routine. Using a deconvolution algorithm the relative spatial arrangement of fine particles, and even of nanoparticles, within RBC was possible.
FIGURE 3. EELS images of TiO2 and silver enhanced gold particles. Ultrafine or small aggregates (1 µm usually remain on the epithelial surface upon their deposition (9, 10, 17) and are subject to clearance by cough, mucociliary transport, and/or phagocytosis by macrophages. Smaller particles, i.e., nanoparticles, can penetrate the membranes of the lung cells rapidly (21, 36-38). In addition, it has been shown for various nanoparticle types, particularly those of
